Design and synthesis of novel disulfide linker based nucleotides as reversible terminators for dna sequencing by synthesis

ABSTRACT

Disclosed herein, inter alfa, are compounds, compositions, and methods of use thereof in the sequencing of a nucleic acid.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a § 371 national stage of PCT InternationalApplication No. PCT/US2016/054236, filed Sep. 28, 2016, which claims thebenefit of U.S. Provisional Applications Nos. 62/257,102, filed Nov. 18,2015, and 62/233,950, filed Sep. 28, 2015, the contents of each of whichare hereby incorporated by reference into the application.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED AS AN ASCII FILE

This application incorporates-by-reference nucleotide and/or amino acidsequences which are present in the file named“180326_88060-A-PCT-US_Sequence_Listing_CAE.txt”, which is 2.74kilobytes in size, and which was created Mar. 26, 2018 in the IBM-PCmachine format, having an operating system compatibility withMS-Windows, which is contained in the text file filed Mar. 26, 2018 aspart of this application.

BACKGROUND

DNA sequencing is a fundamental tool in biological and medical research,and is especially important for the paradigm of personalized medicine.Various new DNA sequencing methods have been investigated with the aimof eventually realizing the goal of the $1,000 genome; the dominantmethod is sequencing by synthesis (SBS) an approach that determines DNAsequences during the polymerase reaction. The currently widely usedhigh-throughput SBS technology (Bentley D R, et al. Nature, 2008, 456,53-59) uses cleavable fluorescent nucleotide reversible terminator (NRT)sequencing chemistry that we developed previously (Ju J et al, 2003,U.S. Pat. No. 6,664,079; Ju J et al. Proc Natl Acad Sci USA, 2006, 103,19635-19640). These cleavable fluorescent NRTs were designed based onthe following rationale: each of the four nucleotides (A, C, G, T) ismodified by attaching a unique cleavable fluorophore to the specificlocation of the base and capping the 3′OH group with a small reversiblemoiety so that they are still recognized by DNA polymerase assubstrates. Thus the cleavable fluorescent. NRTs involve two sitemodifications (Ju J et al, 2003, U.S. Pat. No. 6,664,079; Ju J et al.Proc Natl Acad Sci USA, 2006, 103, 19635-19640): a fluorescent dye toserve as a reporter group on the. base and a small chemical moiety tocap the 3′—OH group to temporarily terminate the polymerase reactionafter nucleotide incorporation for sequence determination. Afterincorporation and signal detection, the fluorophore is cleaved and the3′—OH capping moiety removed to resume the polymerase reaction in thenext cycle. These cleavable fluorescent NRTs have proved to be goodsubstrates for reengineered polymerases and have been used extensivelyin next generation DNA sequencing systems (Ju J et al. Proc. Natl AcadSci USA, 2006, 103, 19635-19640; Bentley D R, et al, Nature, 2008, 456,53-59). Moreover, they enable accurate determination of homopolymersequences, since only one base is identified in each cycle.

To achieve long read length in the SBS strategy it is essential that thecleavable linker be stable during the sequencing reactions, and thatthere are few manipulations and that a long tail is not left on the baseafter the cleavage reaction.

BRIEF SUMMARY OF THE INVENTION

A compound of the formula:

wherein

B is a base;

L¹ is covalent linker;

L² is covalent linker;

R³ is —OH, monophosphate, polyphosphate or a nucleic acid;

R^(4A) is hydrogen, —CH₃, —CX¹ ₃, —CHX¹ ₂, —CH₂X¹, —CN, -Ph, substitutedor unsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl;

R^(4B) is hydrogen, —CH₃, —CX² ₃, —CHX² ₂, —CH₂X², —CN, -Ph, substitutedor unsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl;

R⁵ is a detectable label or anchor moiety;

R⁶ is hydrogen or a polymerase-compatible cleavable moiety;

R⁷ is hydrogen or —OR^(7A), wherein R^(7A) is hydrogen or apolymerase-compatible cleavable moiety; and

X¹ and X² are independently halogen.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. IA-1B. SBS using 3′-O-SS(DTM)-dNTP-SS-Dye (where “DTM” refers tothe Dithiomethyl group). (STEP 1) Addition of a DNA polymerase to theprimed template moiety (only the primer strand is shown above) leads tothe incorporation of a complementary 3′-O-SS(DTM)-dNTP-SS-Dye to the 3′end of a primer with high efficiency and specificity. (STEP 2) Afterwashing away the unincorporated labeled molecules, the detection of theunique label attached to the primer extension product determines theidentity of the incorporated nucleotide. (STEP 3) Addition of TCEP orTHP results in the cleavage of the disulfide bond, and therefore to theremoval of the label on the primer extension product and theregeneration of the 3′—OH on the primer extension product. Therepetition of STEP 1 through STEP 3 allows for continuous DNA sequencedetermination. FIG. 1B: SBS using 3′-O-SS(DTM)-dNTP-SS-“anchors” andcorresponding labeled binding molecules. (STEP 1) Addition of a DNApolymerase to the primed template moiety (only the primer strand isshown above) leads to the incorporation of a complementary3′-O-SS(DTM)-dNTPs-SS(DTM)-“anchor” to the 3′ end of a primer with highefficiency and specificity. (STEP 2) Addition of labeled bindingmolecules to the corresponding primer extension product leads toorthogonal binding of the labeled binding molecules with thecorresponding “anchor” moiety on the base of the primer extensionproduct; after washing away the unbound labeled molecule, the detectionof the unique label attached to the primer extension product determinesthe identity of the incorporated nucleotide. (STEP 3) Addition of TCEPor THP results in the cleavage of the disulfide bond, and therefore tothe removal of the label on the primer extension product and theregeneration of the 3′—OH on the primer extension product. Therepetition of STEP 1 through STEP 3 allows for continuous DNA sequencedetermination. The “Anchor” moiety and the labeled binding moleculeinclude any specifically reactive pair that can form a covalent bond ora stable noncovalent bond. The label can be a fluorescent molecule, aFRET cassette or fluorescent dendrimers.

FIG. 2 . Structures of 3′-O-DTM-dNTPs-SS-Dye(3′-O-t-Butyldithiomethyl-dCTP-5—SS-Alexa488,3′-O-t-Butyldithiomethyl-dUTP-5—SS-R6G,3′-O-t-Butyldithiomethyl-dATP-7—SS-Rox) and3′-O-t-Butyldithiomethyl-dGTP-7—SS-Cy5 (wherein “DTM” refers to theDithiomethyl group).

FIGS. 3A-3E. Structures of 3′-O-DTM-dNTPs-SS-Biotin(3′-O-t-Butyldithiomethyl-dATP-7—SS-Biotin, 3′-O-t-Butyldithiomethyl-dGTP-7—SS-Biotin,3′-O-t-Butyldithiomethyl-dUTP-5—SS-Biotin, 3′-O-t-Butyldithiomethyl-dCTP-5—SS-Biotin) and Cy5 dye labeledstreptavidin as an example tagging moiety (multiple Cy5 dyes can beattached to a single streptavidin molecule to increase detectionsensitivity).

FIGS. 4A-4D. One-color SBS using 3′-O-DTM-dNTP-SS-Biotins and Cy5Labeled Streptavidin. A DNA polymerase incorporation reaction isconducted by using one of the four 3′-O-DTM-dNTP-SS-Biotins (FIGS.3A-3E), followed by the addition of Cy5 labeled streptavidin and imagingto determine DNA sequences as described in STEP 1 through STEP 4 (asshown in FIG. 4A Step 1 and repeated in FIGS. 4A-4D steps 2 to 4). Eachstep consists of three parts: (PART a) Add polymerase and one of thefour 3′-O-DTM-dNTP-SS-Biotins followed by washing; if the addednucleotide is complementary to the nucleotide on the templateimmediately next to the 3′ end of the primer, then the added nucleotidewill incorporate into the primer to produce a DNA extension product thathas a Biotin. (PART b) Add Cy5 Labeled Streptavidin, which will bind tothe Biotin on the DNA extension product. (PART c) After washing away theunbound Cy5 labeled streptavidin, perform imaging to detect the Cy5signal for the identification of the incorporated nucleotide. FollowingSTEP 4, addition of THP to the DNA extension products will cleave thedisulfide bond and regenerate a free 3′—OH group on the 3′ end of theDNA extension products. Sequential repetition of the process, consistingof STEP 1 through STEP 4, followed by THP cleavage, allows continuingsequence determination.

FIG. 5 . Structures of 3′-O-DTM-dNTP-SS-“Anchors”(3′-O-t-Butyldithiomethyl-dATP-7—SS-TCO,3′-O-t-Butyldithiomethyl-dCTP-5—SS-PBA,3′-O-t-Butyldithiomethyl-dGTP-7—SS-Biotin,Butyldithiomethyl-dUTP-5—SS-N3). In this set of nucleotide analogues,four different “anchor” moieties, TCO, PBA, Biotin and Azido groups, areattached to the base of dATP, dCTP, dGTP and dTTP, respectively, throughthe DTM linkage, as shown in this figure.

FIG. 6 . Structures of four-color labeled orthogonal binding molecules(Rox-Labeled Tetrazine, Alexa488-Labeled SHA, Cy5-Labeled Streptavidin,and R6G-Labeled Dibenzocyclooctyne) that bond specifically with the four“anchor” moieties in the nucleotide analogues(3′-O-t-Butyldithiomethyl-dATP-7—SS-TCO,3′-O-t-Butyldithiomethyl-dCTP-5—SS-PBA,3′-O-t-Butyldithiomethyl-dGTP-7—SS-Biotin,3′-O-t-Butyldithiomethyl-dTTP-5—SS-N3) listed in FIG. 4 , as follows:Rox is attached to the Tetrazine (which specifically reacts with TCO);Alexa488 is attached to the SHA (which forms a stable complex with PBA);Cy5 is attached to the Streptavidin (which forms a stable complex withBiotin); and R6G is attached to the Dibenzocyclooctyne (DBCO, whichquickly forms a Triazole moiety with an N₃ group). Thus, each nucleotideanalogue listed in FIG. 5 can be labeled by a unique fluorescent dye.

FIG. 7 . Conjugates or complexes between DNA products produced byincorporating anchor labeled 3′-O-DTM nucleotides(3′-O-t-Butyldithiomethyl-dATP-7—SS-TCO,3′-O-t-Butyldithiomethyl-dCTP-5—SS-PBA,3′-O-t-Butyldithiomethyl-dGTP-7—SS-B iotin,3′-O-t-Butyldithiomethyl-dUTP-5—SS-Azo) with fourcorrespondingly-matched labeled binding molecules (Rox-LabeledTetrazine, Alexa488-Labeled SHA, Cy5-Labeled Streptavidin, andR6G-Labeled Dibenzocyclooctyne). The reaction of the DNA extensionproduct containing four “anchor” moieties on the base (FIG. 5 ) withfour correspondingly-matched labeled binding molecules (FIG. 6 ) leadsto each incorporated nucleotide in the DNA extension product beinglabeled with a unique dye. Thus, Rox will be tethered to the DNAextension product through a specific Tetrazine TCO ligation to formPRODUCT 1; Alexa488 will be tethered to the DNA extension productthrough a stable PBA-SHA complex to form PRODUCT 2; Cy5 will be tetheredto the DNA extension product through a Biotin Streptavidin complex toform PRODUCT 3; and R6G will be tethered to the DNA extension productthrough triazole formation via a click reaction betweenDibenzocyclooctyne and an azido group to form PRODUCT 4.

FIG. 8 . SBS using 3′-O-SS(DTM)-dNTP-SS-“anchors”(3′-O-t-Butyldithiomethyl-dATP-7—SS-TCO,3′-O-t-Butyldithiomethyl-dCTP-5—SS-PBA,3′-O-t-Butyldithiomethyl-dGTP-7—SS-Biotin,3′-O-t-Butyldithiomethyl-dUTP-5—SS-N3) and four correspondingly matcheddye labeled binding molecules (Rox-Labeled Tetrazine, Alexa488-LabeledSHA, Cy5-Labeled Streptavidin, and R6G-Labeled Dibenzocyclooctyne).Addition of the DNA polymerase and the four 3′-O-SS(DTM)-dNTP-SS-anchors(3′-O-DTM-dATP-7—SS-TCO, 3′-O-DTM-dCTP-5—SS-PBA,3′-O-DTM-dGTP-7—SS-Biotin and 3′-O-DTM-dUTP-5—SS-N₃) (FIG. 5 ) to theimmobilized primed DNA template enables the incorporation of thecomplementary nucleotide analogue to the growing DNA strand to terminateDNA synthesis. After washing away the unincorporated nucleotideanalogues, the dye labeled binding molecules (FIG. 6 ) are added; thesewill specifically connect with each of the four unique “anchor” moietieson the DNA extension product to enable the labeling of each DNA productterminated with each of the four nucleotide analogues (A, C, G, T) withthe four distinct fluorescent dyes (FIG. 7 ). Detection of the uniqueflourescence signal from each of the fluorescent dyes on the DNAproducts allows for the identification of the incorporated nucleotide.Next, treatment of the DNA products with THP cleaves the SS linker,leading to the removal of the fluorescent dye and the regeneration of afree 3′—OH group on the DNA extension product, which is ready for thenext cycle of the DNA sequencing reaction (as shown in the subsequentsteps of the figure).

FIGS. 9A-9C. Structures of Fluorescent (Cy5) Dcndrimer ConjugatedTetrazine (FIG. 9A and FIG. 9C) and 3′-O-SS(DTM)-dNTPs-SS-TCO (FIG. 9B).Incorporation of each of the four 3′-O-DTM-dNTP-SS-TCO into the growingDNA strand in the polymerase reaction terminates the DNA synthesis,leading to DNA products that have a TCO group. Coupling of the DNAproducts that have a TCO group with either Molecule A (FIG. 9A) orMolecule B (FIG. 9C) that has the Tetrazine moiety through TCO-Tetrazineligation allows the DNA product to be labeled with multiple fluorescentdyes, thereby facilitating signal amplification for detection to performeither SBS at the single-molecule level or at an ensemble level(following a scheme similar to the one shown in FIGS. 4A-4D)

FIG. 10 . Example of a Peptide-Based Fluorescent (Cy5) DendrimerConjugated Tetrazine (Molecule A) and Polymer Conjugated Tetrazine(Molecule B). Incorporation of each of the four 3′-O-DTM-dNTP-SS-TCOinto the growing DNA strand in the polymerase reaction terminates theDNA synthesis, leading to DNA products that have a TCO group. Couplingof the DNA products that have a TCO group with either Molecule A orMolecule B (shown above) that has a Tetrazine moiety throughTCO-Tetrazine ligation allows the DNA product to be labeled withmultiple fluorescent dyes, thereby facilitating signal amplification fordetection to perform either SBS at the single-molecule level or at anensemble level (following a scheme similar to the one shown in FIG. 4 ).

FIGS. 11A-11D. Examples of FRET Cassette Labeled Binding Molecules. TheFRET cassette strategy provides numerous distinct FRET signal signaturesby altering the distance between donor and acceptor fluorophorcs.Binding molecules conjugated to such FRET cassettes with four uniqueFRET signal signatures enables the coupling of such FRET cassettes tothe DNA extension product using an “anchor” moiety coupling reaction;this allows for the use of two different fluorescent dyes with distinctemissions through FRET to perform scarless 2-color SBS to identify thefour DNA bases. In the set of FRET cassette labeled binding moleculesshown above, Rox and Cy5, serving as donor and acceptor respectively,are attached with 7 or 3 dSpacer monomers to yield two different FRETcassettes: FRET Cassette A (Rox-7-Cy5 attached to SHA), which has a longseparation distance of 7 dSpacer monomers between Rox and Cy5, will havea less efficient energy transfer from the donor (Rox) to the accepter(Cy5), thereby generating a weak Cy5 emission signal and a strong Roxemission signal. FRET Cassette B (Rox-3-Cy5 attached totrans-cyclooctene TCO), which has a short separation distance of 3dSpacer monomers between Rox and Cy5, will have a more efficient energytransfer from the donor (Rox) to the accepter (Cy5), thereby generatinga strong Cy5 signal and a weak Rox signal. In Labeling Molecule C, wherethe single Rox is attached to Tetrazine, only the Rox signal isdetectible. In Labeling Molecule D (FIG. 11D), where the single Cy5 isattached to Streptavidin, only the Cy5 signal is detectible. A schemesimilar to the one indicated in FIG. 8 (four color) is followed toperform SBS by carrying out the following steps to sequence DNA:Addition of the DNA polymerase and the four3′-O-SS(DTM)-dNTP-SS-“anchor” (3′-O-DTM-dATP -7—SS-TCO, 3′-O-DTM-dCTP-5—SS-PBA, 3′-O-DTM-dGTP -7—SS-Biotin and 3′-O-DTM-dTTP-5—SS-N₃) to theimmobilized primed DNA template enables the incorporation of thecomplementary nucleotide analogue to the growing DNA strand to terminateDNA synthesis. After washing away the unincorporated nucleotideanalogues, addition of the dye labeled binding molecules (A, B, C, D),which will specifically connect with each of the four unique “anchor”moieties on each DNA extension product, enables the labeling of each DNAproduct terminated with each of the four nucleotide analogues (A, C, G,T) with four distinct flourescent signatures. Detection of the uniqueflourescent signatures from the labeled DNA products allows for theidentification of the incorporated nucleotide. Next, treatment of theDNA products with THP cleaves the SS linker, leading to the removal ofthe fluorescent label and the regeneration of a free 3′—OH group on theDNA extension product, which is ready for the next cycle of the DNAsequencing reaction.

FIG. 12 . General Scheme of FRET Cassette Labeled Binding Molecules(e.g., SHA, Tetrazine, DBCO, Streptavidin, etc.). The use of FRETcassettes provides numerous distinct FRET signal signatures (A, B, C, D)by altering the distance between the donor and the acceptorfluorophores. A scheme similar to the one indicated in FIG. 8 isfollowed to perform SBS by carrying out the following steps to sequenceDNA: Addition of the DNA polymerase and the four3′-O-SS(DTIVE)-dNIP-SS-“anchor” (3′-O-DTM-dATP-7—SS-TCO, 3′-O-DTM-dCTP-5—SS-PBA, 3′-O-DTM-dGTP-7—SS-B iotin and 3′-O-DTM-dU TP-5—SS-N₃) tothe immobilized primed DNA template enables the incorporation of thecomplementary nucleotide analogue to the growing DNA strand to terminateDNA synthesis. After washing away the unincorporated nucleotideanalogues, addition of the dye labeled binding molecules (A, B, C, D),which will specifically connect with each of the four unique “anchor”moieties on each DNA extension product, enable the labeling of each DNAproduct terminated with each of the four nucleotide analogues (A, C, G,T) with four distinct fluorescent signatures. Detection of the uniquefluorescent signatures from the labeled DNA products allows for theidentification of the incorporated nucleotide. Next, treatment of theDNA products with. THP cleaves the SS linker, leading to the removal ofthe fluorescent label and the regeneration of a free 3′—OH group on theDNA extension product, which is ready for the next cycle of the DNAsequencing reaction.

FIGS. 13A-13J. Example structures of 3′-O-DTM-dNTP-SS-Dyes Rox,3′-O-DTM-dCTP-5—SS-Al exa488); 3′-O-SS(DTM)-dNTP-SS-“anchors” (3′-O-DTM-dGTP-7—SS-TCO and 3′-O-DTM-dUTP-5—SS-N₃), with theircorresponding Dye Labeled Binding Molecules (Rox Labeled Tetrazine andAlexa488 Labeled Dibenzocyclooctyne).

FIGS. 14A-14B. Use of 3′-O-DTM-dNTP-SS-Dyes (3′-O-DTM-dATP-7—SS-Rox,3′-O-DTM-dCTP-5—SS-Alexa488); 3′-O-SS(DTM)-dNTP-SS-“anchors”(3′-O-DTM-dGTP-7—SS-TCO and 3′-O-DTM-clUTP-5—SS-N₃), with theircorresponding Dye Labeled Binding Molecules (Rox Labeled Tetrazine aridAlexa488 Labeled Dibenzocyclooctyne) to perform 2-color DNA SBS.Addition of DNA polymerase and the four nucleotide analogues(3′-O-DTM-dATP-7—SS-Rox, 3′-O-DTM-dCTP-5—SS-Alexa488,3′-O-DTM-dGTP-7—SS-TCO and 3′-O-DTM-dUTP-7—SS-N₃) to the immobilizedprimed DNA template enables the incorporation of the complementarynucleotide analogue to the growing DNA strand to terminate DNA synthesis(STEP I). After washing away the unincorporated nucleotide analogues,the flourescent signal from Rox and BodipyFL is detected to identify theincorporated nucleotide as A (labeled with Rox) and C (labled withBodipyFL). Next, the dye labeled binding molecules (Rox-Tetrazine andBodipyFL-Dibenzocyclooctyne) are added to the DNA extension products(STEP 2), which will specifically connect with the two unique “anchor”moieties (TCO and N3) on each DNA extension product, to enable thelabeling of each DNA product terminated with each of the two nucleotideanalogues (G and T) with two distinct fluorescent dyes (labeled with Roxfor G and labeled with BodipyFL for T). Detection of the unique, newlyproduced fluorescence signal from Rox and BodipyFL on the DNA extensionproducts (in addition to the signal from STEP 1), allows for theidentification of the newly incorporated nucleotides as G and Trespectively. Next, treatment of the DNA products with THP cleaves theSS linker, leading to the removal of the fluorescent dye and theregeneration of a free 3′—OH group on the DNA extension product (STEP3), which is ready for the next cycle of the DNA sequencing reaction (asshown in the subsequent steps of the figure).

FIGS. 15A-15B. Structures of Labeled Binding Molecules conjugated withfluorescent dyes via different cleavable linkers (which are highlightedin parentheses in this Figure). Tetrazine is tethered to ATT0647N via anazo linkage (Tetrazine-Azo(linker)-ATTO647N), which can be cleaved bysodium dithionite (Na₂S₂O₄); Streptavidin is tethered to ATT0647N via adimethylketal linkage (Streptavidin-Dimethylketal(linker)-ATT0647N)),which can be cleaved under weak acidic conditions such as a citric acidbuffer (pH 4); SHA is tethered to ATT0647N via a photocleavablenitrobenzyl linkage (SHA-2-Nitrobenzyl(linker)-ATT0647N), which can becleaved by photoirradiation; DBCO is tethered to ATT0647N via an allyllinkage (Dibenzocyclooctyne-Allyl(linker)-ATTO647N), which can becleaved by Pd(0); DBCO can also be tethered to ATT0647N via Dde linkage(Dibenzocyclooctyne-Dde(linker)-ATT0647N), which can be cleaved byhydrazine. ATT0647N labeled Streptavidin (Streptavidin-ATT0647N) canalso be used in combination with three other binding moleculesconjugated with fluorescent dyes via different cleavable linkers.

FIGS. 16A-16B. Sample Structures of 3′-O-SS(DTM)-dNTP-SS-“anchors”(3′-O-DTM-dATP-7—SS-N3, 3′-O-DTM-dCTP-5—SS-Biotin,3′-O-DTM-dUTP-5—SS-TCO) along with their corresponding Labeled BindingMolecules [DBCO-Azo(—N═N-Linker)-ATTO647N,Tetrazine-Dde(Linker)-ATT0647N, and Streptavidin-ATTO647N] conjugatedwith one fluorescent dye via different cleavable linkers in combinationwith 3′-O-t-Butyl-SS(DTM)-dGTP (3′-O—SS-dGTP) for performing one-colorSBS at the single-molecule level or at the ensemble level.

FIGS. 17A-17B. One color SBS reaction scheme approach 1: (1) In thepresence of DNA polymerase, three nucleotides with anchor[3′-O-DTM-dATP-7—SS-N₃, 3′-O-DTM-dCTP-5—SS-Biotin,3′-O-DTM-dUTP-5—SS-TCO] and 3′-t-Butyl-SS(DTM)-dGTP, as shown in FIG. 16are added to the primed DNA templates to allow incorporation into theprimer; (2) The fluorescent label (ATT0647N, for example) is attached byadding DBCO-Azo-(—N═N-Linker)-ATTO647N, Tetrazine-Dde(Linker)-ATTO647Nand Streptavidin-ATTO647N (as shown in FIG. 16 ) to the DNA extensionproducts that contain the incorporated nucleotide analogues with anchor,which leads to the labeling of all the incorporated nucleotides (exceptG) due to specific anchor-binding molecule interaction; (3) Afterwashing, the first round of imaging is performed, and the DNA productsterminated with A, C and T all display the same color, while the DNAproducts that do not emit a signal are terminated by the nucleotide G;(4) The first cleavage (I) is conducted by treatment with sodiumdithionite (Nu2S₂O₄), which only cleaves the azo linkage to remove thefluorescent dye from the DNA products terminated with the A nucleotide.The second round of imaging is performed. If the fluorescent signaldisappears after cleavage I, the DNA products are determined as havingincorporated an A nucleotide; (5) The second cleavage (II) is conductedby treatment with hydrazine (N₂H₄), which will cleave the Dde linkage toremove the fluorescent dye from the DNA products terminated with the Tnucleotide. The third round of imaging is performed. If the fluorescentsignal disappears after cleavage II, the DNA products are determined ashaving incorporated a T nucleotide. The DNA products with unchangedfluorescent signals arc identified by inference as being terminated by aC nucleotide; (6) The third cleavage (III) is conducted with THP tocleave the disulfide bond and remove the dye on C, so the change of thesignal after the THP treatment verifies the DNA products as beingterminated by a C nucleotide. Meanwhile, the THP treatment will alsocleave the DTM (SS) bond to regenerate free 3′—OH on all the DNAextension products, which are ready for subsequent cycles ofsingle-color DNA SBS. (7) Steps 1 to 6 are repeated to continuesubsequent cycles of single-color DNA SBS.

FIGS. 18A-18B. Sample structures of 3′-O-DTM-dNTP-SS-Dyes(3′-O-DTM-dATP-7—SS-Rox), 3′-O-SS(DTM)-dNTP-SS-“anchors”(3′-O-DTM-dUTP-5-SS-N₃ and 3-O-DTM-dCTP-5—SS-Biotin) along with theircorresponding Labeled Binding Molecules [DBCO-Azo(—N═N-Linker)-Rox andStreptavidin-Rox] conjugated with one fluorescent dye via differentcleavable linkers in combination with 3′-O-t-Butyl-SS(DTM)-dGTP(3′-O—SS-dGTP) for performing one-color SBS at the single-molecule levelor at the ensemble level.

FIGS. 19A-19B. One color SBS reaction scheme approach 2: (1) In thepresence of DNA polymerase, two anchor labeled nucleotides(3′-O-DTM-dUTP-5—SS-N₃ and 3′-O-DTM-dCTP-5—SS-Biotin),3′-O-DTM-dATP-7—SS-Rox and 3′-O-t-Butyl-SS(DTM)-dGTP, as shown in FIG.18 , are added to the primed DNA templates to allow incorporation intothe primer; (2) After washing, the first round of imaging is performed,and the DNA products terminated with an A nucleotide analogue displaythe Rox signal and therefore are determined as having incorporated an Anucleotide, while the other DNA products terminated at G, C, T will notdisplay any fluorescent signals; (3) The fluorescent label (Rox, forexample) is attached to DNA by adding DBCO-Azo-(-N═N-Linker)-Rox andStreptavidin-Rox (as shown in FIG. 18 ) to the DNA extension productsthat contain the incorporated anchor labeled nucleotide analogues, whichleads to the labeling of all the incorporated nucleotides (except G) dueto specific anchor-binding molecule interaction; (4) After washing, thesecond round of imaging is performed, and the DNA products terminatedwith A, C and T all display the same Rox signal, while the DNA productsthat do not emit a signal are terminated by the nucleotide G; (5) Thefirst cleavage (T) is conducted by treatment with sodium dithionite(Na₂S₂O₄), which only cleaves the azo linkage to remove the fluorescentdye Rox from the DNA products terminated with the T nucleotide. Thesecond round of imaging is performed. If the Rox fluorescent signaldisappears after cleavage I, the DNA products are determined as havingincorporated a T nucleotide; (6) The second cleavage (II) is conductedwith THP to cleave the disulfide bond and remove the dye from the DNAextension products terminated with nucleotides A and C, so the change ofthe signal after the THP treatment determines the DNA products as beingterminated by a C nucleotide, because DNA products terminated by an Anucleotide have already being determined in the first round of imagingdescribed above. Meanwhile, the THP treatment will also cleave the DTM(SS) bond to regenerate free 3′—OH on all the DNA extension products,which are ready for subsequent cycles of single-color DNA SBS. Steps 1to 6 are repeated to continue subsequent cycles of single-color DNA SBS.

FIGS. 20A-20B. Sample structures of 3′-O-DTM-dNTP-SS-Dye(3′-O-DTM-dATP-7—SS-Rox), 3′-O-SS(DTM)-dNTP-SS-“anchors”(3′-O-DTM-dUTP—S—SS-TCO, 3′-O-DTM-dCTP—S—SS-Biotin and3′-O-DTM-dGTP-7—SS-N₃) along with their corresponding Labeled BindingMolecules [Tetrazine-Dde(Linker)-Rox, Streptavidin-Rox andDBCO-Azo(-N═N-Linker)-Rox] conjugated with one fluorescent dye viadifferent cleavable linkers for performing one-color SBS at thesingle-molecule level or at the ensemble level.

FIGS. 21A-21F. One color SBS reaction scheme approach 3: (1) In thepresence of DNA polymerase, three anchor labeled nucleotides(3′-O-DTM-dUTP—S—SS-TCO, 3′-O-DTM-dCTP-5—SS-Biotin and3′-O-DTM-dGTP-7—SS-N₃) and 3′-O-DTM-dATP-7—SS-Rox, as shown in FIG. 20are added to the primed DNA templates to allow incorporation into theprimer; (2) After washing, the first round of imaging is performed, andthe DNA products terminated with an A nucleotide analogue display theRox signal and therefore are determined as having incorporated an Anucleotide, while the other DNA products terminated at G, C, T will notdisplay any fluorescent signals; (3) The fluorescent label (Rox, forexample) is attached to DNA by adding DBCO-Azo-(—N═N-Linker)-Rox,Tetrazine-Dde-Rox and Streptavidin-Rox (as shown in FIG. 20 ) to the DNAextension products that contain the incorporated anchor labelednucleotide analogues, which leads to the labeling of all theincorporated nucleotides due to specific anchor-binding moleculeinteraction; (4) After washing, the second round of imaging isperformed, and the DNA products terminated with A, G, T, C all displaythe same Rox signal. Subtraction of the Rox signals from the DNAproducts determined in the first round of imaging as being terminated atan A nucleotide reveals the DNA products terminated at G, T, C; (5) Thefirst cleavage (I) is conducted by treatment with sodium dithionite(Na₂S₂O₄), which only cleaves the azo linkage to remove the fluorescentdye Rox from the DNA products terminated with the G nucleotide. Thesecond round of imaging is performed. If the Rox fluorescent signaldisappears after cleavage I, the DNA products are determined as havingincorporated a G nucleotide; (6) The second cleavage (II) is conductedwith hydrazine (N₂H₄), which will cleave the Dde linkage to remove thefluorescent dye Rox from the DNA products terminated with the Tnucleotide. The third round of imaging is performed. If the Roxfluorescent signal disappears after cleavage II, the DNA products aredetermined as having incorporated a T nucleotide. If the Rox fluorescentsignal stays after cleavage II, the DNA products are determined ashaving incorporated a C nucleotide; (7) The third cleavage (III) isconducted with THP to cleave the disulfide bond and remove the Rox dyefrom the DNA extension products terminated with nuclotides A and C, sothe change of the signal after the THP treatment confirms the DNAproducts as being terminated by a C nucleotide, because DNA productsterminated by an A nucleotide have already being determined in the firstround of imaging described above. Meanwhile, the THP treatment will alsocleave the DTM (SS) bond to regenerate free 3′—OH on all the DNAextension products, which are ready for subsequent cycles ofsingle-color DNA SBS. Steps 1 to 7 are repeated to continue subsequentcycles of single-color DNA SBS.

FIG. 22 . Structures of 3′-O—SS-dNTP-CleavableLinker-Dye(3′-O-DTM-dATP-7—SS-Rox, 3′-O-DTM-dCTP-5-Nitrobenzyl-Rox and3′-O-DTM-dUTP-5-Allyl-Rox) and 3′-O-SS(DTM)-dGTP.

FIGS. 23A-23D. One color SBS reaction scheme approach 4: (1) In thepresence of DNA polymerase, the three 3′-O-DTM-dNTP-CleavableLinker-Dyes(3′-O-DTM-dATP-7—SS-Rox, 3′-O-DTM-dCTP-5-Nitrobenzyl-Rox and3′-O-DTM-dUTP-5-Allyl-Rox) and 3′-O-tButyl-SS-dGTP, as shown in FIG. 22are added to the primed DNA templates to allow incorporation into theprimer; (2) After washing, the first round of imaging is performed, andthe DNA products terminated with C, T and A all display the same Roxsignal, while the DNA products that do not emit a signal are terminatedby the nucleotide G; (3) The first cleavage (I) is conducted byphoto-irradiation at ˜350 nm to remove the fluorescent dye Rox from theDNA products terminated with the C nucleotide. The second round ofimaging is performed. If the Rox fluorescent signal disappears aftercleavage I, the DNA products are determined as having incorporated a Cnucleotide; (4) The second cleavage (II) is conducted with Pd (O), whichwill cleave the allyl linkage to remove the fluorescent dye Rox from theDNA products terminated with the T nucleotide. The third round ofimaging is performed. if the Rox fluorescent signal disappears aftercleavage II, the DNA products are determined as having incorporated a Tnucleotide. If the Rox fluorescent signal remains after cleavage II, theDNA products are determined as having incorporated an A nucleotide; (5)The third cleavage (III) is conducted with THP to cleave the disulfidebond and remove the Rox dye from the DNA extension products terminatedwith nuclotide A, so the change of the signal after the THP treatmentconfirms the DNA products as being terminated by an A nucleotide.Meanwhile, the THP treatment will also cleave the DTM (SS) bond toregenerate free 3′—OH on all the DNA extension products, which are readyfor subsequent cycles of single-color DNA SBS. Steps 1 to 4 are repeatedto continue subsequent cycles of single-color DNA SBS

FIGS. 24A-24B. Structures of Four 3′-O—SS-dNTP-(SS)CleavablcLinkcr-Dyesand 3′-O—SS-dNTP Used for Four Color Sequencing. (Sequencing data usingthese nucleotides are shown in FIG. 32 .)

FIGS. 25A-25F. Four color SBS with chasing. SBS using3′-O-SS(DTM)-dNTP-SS-Dye (3′-O-t-Butyldithiomethyl(SS)-dATP-SS-Rox,3′-O-t-Butyldithiomethyl(SS)-dCTP-SS-Alexa488,3′-O-t-Butyldithiomethyl(SS)-dGTP-SS-Cy5,3′-O-t-Butyldithiomethyl(SS)-dUTP-SS-R6G) (FIG. 24 ) and four3′-O-t-Butyldith om ethyl (SS)-dN'TPs (3′-O-t-Butyldithiomethyl(SS)-dATP, 3′-O-t-Butyldithiomethyl(SS)-dCTP,3′-O-t-Butyldithiomethyl(SS)-dTTP and 3′-O-t-Butyldithiomethyl(SS)-dGTP)(FIG. 24 ). Step 1, Labeling: addition of the DNA polymerase and thefour 3′-O-SS(DTM)-dNTP-SS-Dye (3′-O-t-Butyldithiomethyl(SS)-dATP-SS-Rox,3′-O-t-Butyldithiomethyl(SS)-dCTP-SS-Alexa488,3′-O-t-Butyldithiomethyl(SS)-dGTP-SS-Cy5 and3′-O-t-Butyldithiomethyl(SS)-dUTP-SS-R6G) to the immobilized primed DNAtemplate enables the incorporation of the complementary dye labelednucleotide analogue to the growing DNA strand, the growing DNA strand isterminated with each of the four nucleotide analogues (A, C, G, T) withthe four distinct fluorescent dyes. Step 2, Chase: addition of the DNApolymerase and four 3′-O-t-Butyldithiomethyl(SS)-dNTPs(3′-O-t-Butyldithiomethyl(SS)-dATP, 3′-O-t-Butyldithiomethyl(SS)-dCTP,3′-O-t-Butyldithiomethyl(SS)-dTTP and 3′-O-t-Butyldithiomethyl(SS)-dGTP)to the immobilized primed DNA template enables the incorporation of thecomplementary 3″-O—SS-nucleotide analogue to the growing DNA strandsthat were not extended with one of the dye labeled3′-O-t-Butyldithiomcthyl(SS)-dNTP in step 1. The growing DNA strands arcterminated with one of the four nucleotide analogues (A, C, G, T) withthe four distinct flourescent dyes or the same one of the fournucleotide analogues (A, C, G, T) without dye. After washing away theunincorporated nucleotide analogues (Step 3), detection of the uniquefluorescence signal from each of the fluorescent dyes on the DNAproducts allows for the identification of the incorporated nucleotidefor sequence determination (Step 4). Next, in Step 5 , treatment of theDNA products with THP cleaves the SS linker, leading to the removal ofthe fluorescent dye and the regeneration of a free 3′—OH group on theDNA extension product, which is ready for the next cycle of the DNAsequencing reaction.

FIGS. 26A-26F. Four color SBS without chasing. SBS using3′-O-SS(DTM)-dNTP-SS-Dye (3′-O-t-Butyldithiomethyl(SS)-dATP-SS-Rox. 3LO-t-Butyldithiomethyl (SS)-dCTP-SS-Alexa488,3′-O-t-Butyldithiomethyl(SS)-dGTP-SS-Cy5,3′-O-t-Butyldithiomethyl(SS)-dUTP-SS-R6G) (FIG. 24 ) Step 1, addition ofthe DNA polymerase and the four 3′-O-SS(DTM)-dNTP-SS-Dye(3′-O-t-Butyldithiomethyl(SS)-dATP-SS-Rox, 3′-O-t-Butyldithiomethyl(SS)-dC'TP-SS-A 1 exa488, 3′-O-t-Butyldithiomethyl(SS)-dGTP-SS-Cy5,3′-O-t-Butyldithiomethyl(SS)-dUTP-SS-R6G) to the immobilized primed DNAtemplate enables the incorporation of the complementary nucleotideanalogue to the growing DNA strand. The growing DNA strand is terminatedwith each of the four nucleotide analogues (A, C, G, T) with the fourdistinct fluorescent dyes. After washing (Step 2) to removeunincorporated dye labeled nucleotide analogues, detection of the uniquefluorescence signal (Step 3) from each of the fluorescent dyes on theDNA products allows for the identification of the incorporatednucleotide. Next, in Step 4, treatment of the DNA products with THPcleaves the SS linker, leading to the removal of the fluorescent dye andthe regeneration of a free 3′—OH group on the DNA extension product,which is ready for the next cycle of the DNA sequencing reaction.

FIGS. 27A-27B. Four color SBS with a mixture of labeled and unlabeledreversible terminators. SBS using 3′-O-SS (DTM)-dNTP-SS-Dye(3′-O-t-Butyldithiomethyl(SS)-dATP-SS-Rox,3′-O-t-Butyldithiomethyl(SS)-dCTP-SS-Alexa488, 3′-O-t-Butyldithiomethyl(SS)-dGTP-SS-Cy5, 3 ′-O-t-Butyldithiomethyl(SS)-dUTP-SS-R6G) (FIG. 24 )and four 3′-O-t-Butyldithiomethyl(SS)-dNTPs(3′-O-t-Butyldithiomethyl(SS)-dATP, 3′-O-t-Butyldithiomethyl(SS)-dCTP,3′-O-t-Butyldithiomethyl(SS)-dTTP and 3′-O-t-Butyldithiomethyl(SS)-dGTP)(FIG. 24 ). Step 1, Addition of the DNA polymerase, the four3′-O-SS(DTM)-dNTP-SS-Dye ((3′-O-t-Butyldithiomethyl(SS)-dATP-SS-Rox,3′-O-t-Butyldithiomethyl(SS)-dCTP-SS-Alexa488,3′-O-t-Butyldithiomethyl(SS)-dGTP-SS-Cy5,3′-O-t-Butyldithiomethyl(SS)-dUTP-SS-R6G) and four3′-O-t-Butyldithiomethyl(SS)-dNTPs (3′4)4-Butyldithiomethyl(SS)-dATP, 3′-O-t-Butyldithiomethyl (SS)-dCTP, 3′-O-t-Butyldithiomethyl(SS)-dTTP and3′-O-t-Butyldithiomethyl(SS)-dGTP) to the immobilized primed DNAtemplate enables the incorporation of the complementary nucleotideanalogue to the growing DNA strand, The growing DNA strand is terminatedwith each of the four nucleotide analogues (A, C, G, T) with the fourdistinct fluorescent dyes or without dye labeling. After washing away(Step 2) the unincorporated nucleotide analogues, detection of theunique fluorecence signal (Step 3) from each of the fluorescent dyes onthe DNA products allows for the identification of the incorporatednucleotide. Next, in Step 4, treatment of the DNA products with THPcleaves the SS linker, leading to the removal of the fluorescent dye andthe regeneration of a free 3′—OH group on the DNA extension product,which is ready for the next cycle of DNA sequencing.

FIGS. 28A-28B. Four color SBS with chasing and walking In this example,the four fluorescent nucleotide analogues with 3′-O-blocking groups aswell as the four unlabeled blocked nucleotides are added together toobtain sequencing as in the previous example (FIG. 27 ). Next, the DNAis denatured to strip off the extended primer. The original primer isreannealed to the DNA template. After this, three natural nucleotidesand one 3′-O-blocked nucleotide, all unlabeled, are added to theoriginal primed template to carry out extension to the positioncomplementary to the next occurrence of the single 3′-O-blockednucleotide. This is repeated a sufficient number of times to rapidlywalk to approximately the position where the prior sequencing run hadended (the number of steps in the walk will be determined by theexpected base frequencies in the genome of interest). At this point thefour labeled and four unlabeled 3′-O-blocked nucleotides are added toproduce a new sequence read that extends from where the previous readleft off. The sequence-denature-walk protocol can be repeated severaltimes to obtain much longer sequences than could be obtained withoutwalking.

FIGS. 29A-29H. MALDI-TOF MS spectra showing single base extension andcleavage products using 3′—SS-dATP-SS-Rox(M.W. 1344),3′—SS-dCTP-SS-Alexa488(M.W.1319), 3′—SS-dGTP-SS-Cy5(M.W. 1482), and3′—SS-dUTP-SS-R6G(M.W. 1233) (structures shown in FIG. 24 ),respectively. The masses of the expected extension products are 7253,6288, 7438, and 6221 Daltons, respectively. The masses of the expectedcleavage products are 6451, 5488, 6515, and 5508 Daltons. The y axisshows the percent intensity, while the x axis shows mass (m/z).

FIGS. 30A-30D. Example structures and Experimental scheme of continuousDNA sequencing by synthesis (left) using four 3′-O-Et-dithiomethyl-dNTPsreversible terminators (3′-O—SS-Et-dNTPs or 3′-O-DTM-dNTPs) andMALDI-TOF MS spectra (right) obtained from each step of extension andcleavage. THP =(tris(hydroxypropyl)phosphine). The masses of theexpected extension products are 4381, 4670, 4995, and 5295 Darespectively. The masses of the expected cleavage products are 4272,4561, 4888, and 5186 Da. The measured masses shown in FIG. 30B arewithin the resolution of MALDI-TOF MS. Experimental scheme of continuousDNA sequencing by synthesis (left) using four 3′-O-t-Bu-SS-dNTPsreversible terminators (FIG. 30C) and MALDI-TOF MS spectra FIG. D)obtained from each step of extension and cleavage. The masses of theexpected extension products are 4404, 4697, 5024, and 5328 Daltonsrespectively. The masses of the expected cleavage products are 4272,4563, 4888, and 5199 Daltons.

FIG. 31 . Demonstration of walking strategy. The DNA template (SEQ IDNO:1) and primer (SEQ ID NO:2) shown above were used (the portion of thetemplate shown in green is the primer binding region) and incubation wascarried out using Therminator IX DNA polymerase, dATP, dCTP, dTTP and3′-O-t-butyl-SS-dGTP. After the first walk, the primer was extended tothe point of the next C in the template (rightmost C highlighted in redin the template strand). The size of the extension product was 5330Daltons (5328 Da expected) as shown in the top left MALDI-TOF MS trace.After cleavage with THP, the 5198 Da product shown at the top right wasobserved (5194 Da expected). A second walk was performed withTherminator IX DNA polymerase, dATP, dCTP, dTTP and 3′-O-t-butyl-SS-dGTPto obtain the product shown in the middle left trace (7771 Da observed,7775 Da expected to reach the middle C highlighted in red). Aftercleavage, a product of 7643 Da was obtained (expected 7641 Da). Finallya third walk and cleavage were performed, giving products of 9625 Da(9628 Da expected for the leftmost red highlighted C) and 9513 Da (9493Da expected), respectively. This demonstrates the ability to use the3′-O-t-butyl-SS- nucleotide as a terminator for walking reactions. Thesecan be incorporated into a combined sequencing/walking scheme such asthe one depicted in FIG. 27 .

FIG. 32 . Four Color SBS Data on a Solid Surface. A four-colorsequencing data plot of raw fluorescence emission intensity obtained byusing a mixture of 3′—SS-dATP-SS-Rox, 3′—SS-dCTP-SS-Alexa488,3′—SS-dGTP-SS-Cy5, and 3′—SS-dTTP-SS-R6G for each sequencing cycle usingthe self-priming DNA template shown above covalently attached to a glassslide.

FIG. 33 . General structures of derivatives of 3′-O—SS-dNTPs.

FIG. 34 . General Structures of derivatives of 3′-O—SS-dNTPs.

FIGS. 35A-35C. Base-Labeled Reversible Terminators with differentblocking group modifications.

FIG. 36 . Unlabeled Reversible Terminators with different blocking groupmodifications at 3′-O position (used for sequencing, chasing andwalking).

FIG. 37 . Synthesis of 3′-O-DTM-dCTP-5-Nitrobenzyl-Rox.

FIG. 38 . Synthesis of 3′-O-DTM-dUTP—S—Allyl-Rox.

FIG. 39 . Synthesis of 3′O-DTM-dNTP-Nitrobenzyl-R; R refers to Dye or“Anchor” molecule.

FIG. 40 . Synthesis of 3′-O-DTM-dNTP-Allyl-R; R refers to Dye or“Anchor” molecule.

FIG. 41 . Synthesis of 3′-O-DTM-dNTP-Azo(—N═N-Linker)-R; R refers to Dyeor “Anchor” molecule.

FIG. 42 . Synthesis of 3′-O-DTM-dNTP-Dde Linker-R; R refers to Dye or“Anchor” molecule.

FIG. 43 . Synthetic scheme for the preparation of 3′-O-DTM-dNTPs-SS-Dye.

FIG. 44 . Structures of four5(7)-aminopropynyl-3′-O-tBu-dithiomethyl-dNTPs (PA-3′-O-DTM-dNTPs).

FIG. 45 . Structures of four 3′-O-alkyldithiomethyl-dNTPs-SS-Linker-Dye(3′-O-DTM-dNTPs-SS-Dye).

FIG. 46 . Experimental scheme of consecutive extensions (Top) using3′-O-t-Bu-SS-dCTP-SS-BodipyFL reversible terminator and MALDI-TOF MSspectra of the first extension product (Product 1, left, expected MW.6334), the first cleavage product (Product 2, middle, expected MW.5556), and the second extension product (Product 3, right, expected MW.6746).

FIG. 47 . Scheme for synthesis of 3′-O-ethyldithiomethyl-dTTP (7a).

FIG. 48 . Scheme for synthesis of 3′-O-ethyldithiomethyl-dATP (8c).

FIG. 49 . Scheme for synthesis of 3′-O-ethyldithiomethyl-dCTP(3′-O-DTM-dCTP 7d).

FIG. 50 . Scheme for synthesis of5-(3-aminopropynyl)-3′-O-t-butyldithiomethyl-dCTP (5-PA-3′-O-DTM-dCTP).

DETAILED DESCRIPTION

I. Definitions

The abbreviations used herein have their conventional meaning within thechemical and biological arts. The chemical structures and formulae setforth herein are constructed according to the standard rules of chemicalvalency known in the chemical arts.

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they equally encompass thechemically identical substituents that would result from writing thestructure from right to left, e.g., —CH₂O— is equivalent to —OCH₂—.

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a straight (i.e., unbranched) or branchedcarbon chain (or carbon), or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include mono-, di- andmultivalent radicals, having the number of carbon atoms designated(i.e., C₁-C₁₀ means one to ten carbons). Alkyl is an uncyclized chain.Examples of saturated hydrocarbon radicals include, but are not limitedto, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl,isobutyl, sec-butyl, (cyclohexyl)methyl, homologs and isomers of, forexample, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Anunsaturated alkyl group is one having one or more double bonds or triplebonds. Examples of unsaturated alkyl groups include, but are not limitedto, vinyl, 2-propcnyl, crotyl, 2-isopentenyl, 2-(butadicnyl),2,4-pentadienyl, 3-(1,4-pcntadicnyl), ethynyl, 1- and 3-propynyl,3-butynyl, and the higher homologs and isomers. An alkoxy is an alkylattached to the remainder of the molecule via an oxygen linker (—O—). Analkyl moiety may be an alkenyl moiety. An alkyl moiety may be an alkynylmoiety. An alkyl moiety may be fully saturated. An alkenyl may includemore than one double bond and/or one or more triple bonds in addition tothe one or more double bonds. An alkynyl may include more than onetriple bond and/or one or more double bonds in addition to the one ormore triple bonds.

The term “alkylene,” by itself or as part of another substituent, means,unless otherwise stated, a divalent radical derived from an alkyl and anunsaturated alkyl, as exemplified, but not limited by, —CH₂CH₂CH₂CH₂—.Typically, an alkyl (or alkylene) group will have from 1 to 24 carbonatoms, with those groups having 10 or fewer carbon atoms being preferredherein. A “lower alkyl” or “lower alkylene” is a shorter chain alkyl oralkelyene (e.g., alkylene, alkenylenc, or alkynylene) group, generallyhaving eight or fewer carbon atoms. The term “alkenylene,” by itself oras part of another substituent, means, unless otherwise stated, adivalent radical derived from an alkene. The term “alkynylene” by itselfor as part of another substituent, means, unless otherwise stated, adivalent radical derived from an alkyne.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcombinations thereof, including at least one carbon atom and at leastone heteroatom (e.g., O, N, P, Si, and S), and wherein the nitrogen andsulfur atoms may optionally be oxidized, and the nitrogen heteroatom mayoptionally be quaternized. The heteroatom(s) (e.g., O, N, S, Si, or P)may be placed at any interior position of the heteroalkyl group or atthe position at which the alkyl group is attached to the remainder ofthe molecule. Heteroalkyl is an uncyclized chain Examples include, butare not limited to: —CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃,—CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂, —S(O)—CH₃,—CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃,—CH═CH—N(CH₃)—CH₃,—O—CH₃,—O—CH₂—CH₃, and —CN. Up to two or threeheteroatoms may be consecutive, such as, for example, —CH₂—NH—OCH₃ and—CH₂—O—Si(CH₃)₃. A heteroalkyl moiety may include one heteroatom (e.g.,O, N, S, Si, or P). A heteroalkyl moiety may include two optionallydifferent heteroatoms (e.g., O, N, S, Si, or P). A heteroalkyl moietymay include three optionally different heteroatoms (e.g., O, N, S, Si,or P). A heteroalkyl moiety may include four optionally differentheteroatoms (e.g., O, N, S, Si, or P). A heteroalkyl moiety may includefive optionally different heteroatoms (e.g., O, N. S, Si, or P). Aheteroalkyl moiety may include up to 8 optionally different heteroatoms(e.g., O, N, S, Si, or P). The term “heteroalkenyl,” by itself or incombination with another term, means, unless otherwise stated, aheteroalkyl including at least one double bond. A heteroalkenyl mayoptionally include more than one double bond and/or one or more triplebonds in additional to the one or more double bonds. The term“heteroalkynyl” by itself or in combination with another term, means,unless otherwise stated, a heteroalkyl including at least one triplebond. A heteroalkynyl may optionally include more than one triple bondand/or one or more double bonds in additional to the one or more triplebonds.

Similarly, the term “heteroalkylene,” by itself or as part of anothersubstituent, means, unless otherwise stated, a divalent radical derivedfrom heteroalkyl, as exemplified, but not limited by,—CH₂—CH₂—S—CH₂—CH₂— and —CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkelyenegroups, heteroatoms can also occupy either or both of the chain termini(e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, andthe like). Still further, for alkelyene (e.g., alkylene, alkenylene, oralkynylene) and heteroalkelyene linking groups, no orientation of thelinking group is implied by the direction in which the formula of thelinking group is written. For example, the formula —C(O)₂R′— representsboth —C(O)₂R′— and —R′C(O)₂—. As described above, heteroalkyl groups, asused herein, include those groups that are attached to the remainder ofthe molecule through a heteroatom, such as —C(O)R′, —C(O)NR′, —NR′R″,—OR′, —SR′, and/or —SO₂R′. Where “heteroalkyl” is recited, followed byrecitations of specific heteroalkyl groups, such as —NR′R″ or the like,it will be understood that the terms heteroalkyl and —NR′R″ are notredundant or mutually exclusive. Rather, the specific heteroalkyl groupsare recited to add clarity. Thus, the term “heteroalkyl” should not beinterpreted herein as excluding specific heteroalkyl groups, such as—NR′R″ or the like.

The terms “cycloalkyl” and “heterocycloalkyl,” by themselves or incombination with other terms, mean, unless otherwise stated, cyclicversions of “alkyl” and “heteroalkyl,” respectively. Cycloalkyl andheterocycloalkyl are not aromatic. Additionally, for heterocycloalkyl, aheteroatom can occupy the position at which the heterocycle is attachedto the remainder of the molecule. Examples of cycloalkyl include, butare not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,1-cyclohexenyl, 3-cyclohexenyl. cycloheptyl, and the like. Examples ofheterocycloalkyl include, but are not limited to,1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like. A “cycloalkylene” and a“heterocycloalkylene,” alone or as part of another substituent, means adivalent radical derived from a cycloalkyl and heterocycloalkyl,respectively.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“halo(C₁-C₄)alkyl” includes, but is not limited to, fluoromethyl,difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl,3-bromopropyl, and the like.

The term “acyl” means, unless otherwise stated, —C(O)R where R is asubstituted or unsubstituted alkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl.

The term “aryl” means, unless otherwise stated, a polyunsaturated,aromatic, hydrocarbon substituent, which can be a single ring ormultiple rings (preferably from 1 to 3 rings) that are fused together(i.e., a fused ring aryl) or linked covalently. A fused ring aryl refersto multiple rings fused together wherein at least one of the fused ringsis an aryl ring. The term “heteroaryl” refers to aryl groups (or rings)that contain at least one heteroatom such as N. O, or S, wherein thenitrogen and sulfur atoms are optionally oxidized, and the nitrogenatom(s) are optionally quatemized. Thus, the term “heteroaryl” includesfused ring heteroaryl groups (i.e., multiple rings fused togetherwherein at least one of the fused rings is a heteroaromatic ring). A5,6-fused ring heteroarylene refers to two rings fused together, whereinone ring has 5 members and the other ring has 6 members, and wherein atleast one ring is a heteroaryl ring. Likewise, a 6,6-fused ringheteroarylene refers to two rings fused together, wherein one ring has 6members and the other ring has 6 members, and wherein at least one ringis a heteroaryl ring. And a 6,5-fused ring heteroarylene refers to tworings fused together, wherein one ring has 6 members and the other ringhas 5 members, and wherein at least one ring is a heteroaryl ring. Aheteroaryl group can be attached to the remainder of the moleculethrough a carbon or heteroatom. Non-limiting examples of aryl andheteroaryl groups include phenyl, naphthyl, pyrrolyl, pyrazolyl,pyridazinyl, triazinyl, pyrimidinyl, imidazolyl, pyrazinyl, purinyl,oxazolyl, isoxazolyl, thiazolyl, furyl, thienyl, pyridyl, pyrimidyl,benzothiazolyl, benzoxazoyl benzimidazolyl, benzofuran, isobenzofuranyl,indolyl, isoindolyl, benzothiophenyl, isoquinolyl, quinoxalinyl,quinolyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl,3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl,2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl,4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl,2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl,5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl,3-quinolyl, and 6-quinolyl. Substituents for each of the above notedaryl and heteroaryl ring systems are selected from the group ofacceptable substituents described below. An “arylene” and a“heteroarylene,” alone or as part of another substituent, mean adivalent radical derived from an aryl and heteroaryl, respectively. Aheteroaryl group substituent may be —O— bonded to a ring heteroatomnitrogen.

Spirocyclic rings are two or more rings wherein adjacent rings areattached through a single atom. The individual rings within spirocyclicrings may be identical or different. Individual rings in spirocyclicrings may be substituted or unsubstituted and may have differentsubstituents from other individual rings within a set of spirocyclicrings. Possible substituents for individual rings within spirocyclicrings are the possible substituents for the same ring when not part ofspirocyclic rings (e.g. substituents for cycloalkyl or heterocycloalkylrings). Spirocylic rings may be substituted or unsubstituted cycloalkyl,substituted or unsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkyl or substituted or unsubstituted heterocycloalkelyeneand individual rings within a spirocyclic ring group may be any of theimmediately previous list, including having all rings of one type (e.g.all rings being substituted heterocycloalkelyene wherein each ring maybe the same or different substituted heterocycloalkylene). Whenreferring to a spirocyclic ring system, heterocyclic spirocyclic ringsmeans a spirocyclic rings wherein at least one ring is a heterocyclicring and wherein each ring may be a different ring. When referring to aspirocyclic ring system, substituted spirocyclic rings means that atleast one ring is substituted and each substituent may optionally bedifferent.

The symbol “

” denotes the point of attachment of a chemical moiety to the remainderof a molecule or chemical formula.

The term “oxo,” as used herein, means an oxygen that is double bonded toa carbon atom.

The term “alkylarylene” as an arylene moiety covalently bonded to analkelyene (e.g., alkylene, alkenylene, or alkynylene) moiety (alsoreferred to herein as an alkelyene). In embodiments, the alkylarylenegroup has the formula:

An alkylarylene moiety may be substituted (e.g., with a substituentgroup) on the alkelyene (e.g., alkylene, alkenylene, or alkynylene)moiety or the arylene linker (e.g. at carbons 2, 3, 4, or 6) withhalogen, oxo, —N₃, —CF₃, —CCl₃, —CBr₃, —Cl₃, —CN, —CHO, —OH, —NH₂,—COOH, —CONH₂, —NO₂, —SH, —SO₂CH₃—SO₃H, —OSO₃H, —SO₂NH₂, —NHNH₂, —ONH₂,—NHC(O)NHNH₂, substituted or unsubstituted C₁-C₅ alkyl or substituted orunsubstituted 2 to 5 membered heteroalkyl). In embodiments, thealkylarylene is unsubstituted.

Each of the above terms (e.g., “alkyl,” “heteroalkyl,” “cycloalkyl,”“heterocycloalkyl,” “aryl,” and “heteroaryl”) includes both substitutedand unsubstituted forms of the indicated radical. Preferred substituentsfor each type of radical are provided below.

Substituents for the alkyl and heteroalkyl radicals (including thosegroups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkvl, cycloalkenyl, andheterocycloalkenyl) can be one or more of a variety of groups selectedfrom, but not limited to, —OR′, 50 O, ═NR′, ═N—OR′, —NR′R″, —SR,-halogen, —SiR′R″R′″, —OC(O)R, —C(O)R, —CO₂R′, —CONR′R″, —OC(O)NR′R″,—NR″C(O)R′, —NR′—C(O)NR′R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″,—NR—C(NR′R″)═NR′″, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —NR′NR″R″″,—ONR′R″, —NR′C(O)NR″NR′″R″″, —CN, —NO₂, —NR′SO₂R″, —NRC(O)R″,—NR′C(O)—OR″, —NR′OR″, in a number ranging from zero to (2m′+1), wherem′ is the total number of carbon atoms in such radical. R, R′, R″, R′″,and R″″ each preferably independently refer to hydrogen, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl (e.g., aryl substituted with 1-3 halogens),substituted or unsubstituted heteroaryl, substituted or unsubstitutedalkyl, alkoxy, or thioalkoxy groups, or arylalkyl groups. When acompound described herein includes more than one R group, for example,each of the R groups is independently selected as are each R′, R″, R′″,and R″″ group when more than one of these groups is present. When R′ andR″ are attached to the same nitrogen atom, they can be combined with thenitrogen atom to form a 4-, 5-, 6-, or 7-membered ring. For example,—NR′R″ includes, but is not limited to, 1-pyrrolidinyl and4-morpholinyl. From the above discussion of substituents, one of skillin the art will understand that the term “alkyl” is meant to includegroups including carbon atoms bound to groups other than hydrogengroups, such as haloalkyl (e.g., —CF₃ and —CH₂CF₃) and acyl (e.g.,—C(O)CH₃,—C(O)CF₃,—C(O)CH₂OCH₃, and the like).

Similar to the substituents described for the alkyl radical,substituents for the aryl and heteroaryl groups are varied and areselected from, for example: —OR′, —NR′R″, —SR′, -halogen, —SiR′R″R′″,—OC(O)R′, —C(O)R, —COR′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′,—NR′—C(O)NR″R′″, —NR″C(O)₂R′, —NR—C(NR′R″R′″)═NR″″, —NR—C(NR′R″)═NR′″,—S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —NRSO₂R′, —NR′NR″R′″, —ONR′R″,—NR′C(O)NR″NR′″R″″, —CN, —NO₂, —R′, —N₃, —CH(Ph)₂, fluoro(C₁-C₄)alkoxy,and fluoro(C₁-C₄)alkyl, —NR′SO₂R″, —NRC(O)R″, —NR′C(O)-OR″, —NR′OR″, ina number ranging from zero to the total number of open valences on thearomatic ring system; and where R′, R″, R′″, and R″″ are preferablyindependently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl. When a compound described herein includes more than one Rgroup, for example, each of the R groups is independently selected asare each R′, R″, R′″, and R″″ groups when more than one of these groupsis present.

Substituents for rings (e.g. cycloalkyl, heterocycloalkyl, aryl,heteroaryl, cycloalkylene, heterocycloalkylene, arylene, orheteroarylene) may be depicted as substituents on the ring rather thanon a specific atom of a ring (commonly referred to as a floatingsubstituent). In such a case, the substituent may be attached to any ofthe ring atoms (obeying the rules of chemical valency) and in the caseof fused rings or spirocyclic rings, a substituent depicted asassociated with one member of the fused rings or spirocyclic rings (afloating substituent on a single ring), may be a substituent on any ofthe fused rings or spirocyclic rings (a floating substituent on multiplerings). When a substituent is attached to a ring, but not a specificatom (a floating substituent), and a subscript for the substituent is aninteger greater than one, the multiple substituents may be on the sameatom, same ring, different atoms, different fused rings, differentspirocyclic rings, and each substituent may optionally be different.Where a point of attachment of a ring to the remainder of a molecule isnot limited to a single atom (a floating substituent), the attachmentpoint may be any atom of the ring and in the case of a fused ring orspirocyclic ring, any atom of any of the fused rings or spirocyclicrings while obeying the rules of chemical valency. Where a ring, fusedrings, or spirocyclic rings contain one or more ring heteroatoms and thering, fused rings, or spirocyclic rings are shown with one more floatingsubstituents (including, but not limited to, points of attachment to theremainder of the molecule), the floating substituents may be bonded tothe heteroatoms. Where the ring heteroatoms are shown bound to one ormore hydrogens (e.g. a ring nitrogen with two bonds to ring atoms and athird bond to a hydrogen) in the structure or formula with the floatingsubstituent, when the heteroatom is bonded to the floating substituent,the substituent will be understood to replace the hydrogen, whileobeying the rules of chemical valency.

Two or more substituents may optionally be joined to form aryl,heteroaryl, cycloalkyl, or heterocycloalkyl groups. Such so-calledring-forming substituents are typically, though not necessarily, foundattached to a cyclic base structure. In one embodiment, the ring-formingsubstituents are attached to adjacent members of the base structure. Forexample, two ring-forming substituents attached to adjacent members of acyclic base structure create a fused ring structure. In anotherembodiment, the ring-forming substituents are attached to a singlemember of the base structure. For example, two ring-forming substituentsattached to a single member of a cyclic base structure create aspirocyclic structure. In yet another embodiment, the ring-formingsubstituents arc attached to non-adjacent members of the base structure.

Two of the substituents on adjacent atoms of the aryl or heteroaryl ringmay optionally form a ring of the formula -T-C(O)—(CRR′)_(q)—U—, whereinT and U are independently —NR—, —O—, —CRR′—, or a single bond, and q isan integer of from 0 to 3. Alternatively, two of the substituents onadjacent atoms of the aryl or heteroaryl ring may optionally be replacedwith a substituent of the formula -A-(CH₂)_(r)—B—, wherein A and B areindependently —CRR′—, —O—, —NR—, —S—, —S(O)—, —S(O)₂—, —S(O)₂NR′—, or asingle bond, and r is an integer of from 1 to 4. One of the single bondsof the new ring so formed may optionally be replaced with a double bond.Alternatively, two of the substituents on adjacent atoms of the aryl orheteroaryl ring may optionally be replaced with a substituent of theformula —(CRR′)_(s)—X′— (C″R″R′″)_(d)—, where s and d are independentlyintegers of from 0 to 3, and X′ is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or—S(O)₂NR′—. The substituents R, R′, R″, and R′″ are preferablyindependently selected from hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, and substituted or unsubstitutedheteroaryl.

As used herein, the terms “heteroatom” or “ring heteroatom” are meant toinclude boron (B), oxygen (O), nitrogen (N), sulfur (S), phosphorus (P),and silicon (Si).

A “substituent” or “substituent group,” as used herein, means a groupselected from the following moieties:

(A) oxo, halogen, —CF₃, —CHF₂, —CH₂F, —C(halogen)₃, —CH(halogen)₂,—CH₂(halogen), —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃₃H, —SO₄H,—SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHS O₂H, —NHC (O)H,—NHC (O) OH, —NHOH, —OCF₃, —OCHF₂, —OCH₂F, —OCF₃, —OCHF₂, —OCH₂F,—OC(halogen)₃, —OCH(halogen)₂, —OCH₂(halogen), unsubstituted alkylunsubstituted heteroalkyl unsubstituted cycloalkyl, unsubstitutedheterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and

(B) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl,substituted with at least one substituent selected from:

(i) oxo, halogen, —CF₃, —CHF₂, —CH₂F, —C(halogen)₃, —CH(halogen)₂,—CH₂(halogen), -CN. —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H,—SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H,NHC(O)OH, —NHOH, —OCF₃, —OCHF₂, —OCH₂F, —OCF₃, —OCHF₂, —OCH₂F,—OC(halogen)₃, —OCH(halogen)₂, —OCH₂(halogen), unsubstituted alkyl,unsubstituted heteroalkyl, unsubstituted cycloalkyl, un substitutedheterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and (ii)alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl,substituted with at least one substituent selected from:

(a) oxo, halogen, —CF₃, —CHF₂, —CH₂F, —C(halogen)₃, —CH(halogen)₂,—CH₂(halogen), —CN, —OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO41-1,—SO₂NH₂, —NHNH₂, —ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H,—NHC(O)OH, —NHOH, —OCF₃, —OCHF₂, —OCH₂F, —OCF₃, —OCHF₂, —OCH₂F,—OC(halogen)₃, —OCH(halogen)₂, —OCH₂(halogen), unsubstituted alkyl,unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstitutedheterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and

(b) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl,substituted with at least one substituent selected from: oxo, halogen,—CF₃, —CHF₂, —CH₂F, —C(halogen)₃, —CH(halogen)₂, —CH₂(halogen), —CN,—OH, —NH₂, —COOH, —CONH₂, —NO₂, —SH, —SO₃H, —SO₄H, —SO₂NH₂, —NHNH₂,—ONH₂, —NHC(O)NHNH₂, —NHC(O)NH₂, —NHSO₂H, —NHC(O)H, NHC(O)OH, —NHOH,—OCF₃, —OCHF₂, —OCH₂F, —OCF₃, —OCHF₂, —OCH₂F, —OC(halogen)₃,—OCH(halogen)₂, —OCH₂(halogen), unsubstituted alkyl, unsubstitutedheteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl,unsubstituted aryl, unsubstituted heteroaryl.

A “size-limited substituent” or “ size-limited substituent group,” asused herein, means a group selected from all of the substituentsdescribed above for a “substituent group,” wherein each substituted orunsubstituted alkyl is a substituted or unsubstituted C₁-C₂₀ alkyl, eachsubstituted or unsubstituted heteroalkyl is a substituted orunsubstituted 2 to 20 membered heteroalkyl, each substituted orunsubstituted cycloalkyl is a substituted or unsubstituted C₃-C₈cycloalkyl, each substituted or unsubstituted hctcrocycloalkyl is asubstituted or unsubstituted 3 to 8 membered hctcrocycloalkyl, eachsubstituted or unsubstituted aryl is a substituted or unsubstitutedC₆-C₁₀ aryl, and each substituted or unsubstituted heteroaryl is asubstituted or unsubstituted 5 to 10 membered heteroaryl.

A “lower substituent” or “ lower substituent group,” as used herein,means a group selected from all of the substituents described above fora “substituent group,” wherein each substituted or unsubstituted alkylis a substituted or unsubstituted C₁-C₈ alkyl, each substituted orunsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8membered heteroalkyl, each substituted or unsubstituted cycloalkyl is asubstituted or unsubstituted C₃-C₇ cycloalkyl, each substituted orunsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7membered heterocycloalkyl, each substituted or unsubstituted aryl is asubstituted or unsubstituted C₆-C₁₀ aryl, and each substituted orunsubstituted heteroaryl is a substituted or unsubstituted 5 to 9membered heteroaryl.

In some embodiments, each substituted group described in the compoundsherein is substituted with at least one substituent group. Morespecifically, in some embodiments, each substituted alkyl, substitutedheteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl,substituted aryl, substituted heteroaryl, substituted alkylene,substituted heteroalkylene, substituted cycloalkylene, substitutedheterocycloalkylene, substituted arylene, and/or substitutedheteroarylene described in the compounds herein are substituted with atleast one substituent group. In other embodiments, at least one or allof these groups are substituted with at least one size-limitedsubstituent group. in other embodiments, at least one or all of thesegroups are substituted with at least one lower substituent group.

In other embodiments of the compounds herein, each substituted orunsubstituted alkyl may be a substituted or unsubstituted C₁-C₂₀ alkyl,each substituted or unsubstituted heteroalkyl is a substituted orunsubstituted 2 to 20 membered heteroalkyl, each substituted orunsubstituted cycloalkyl is a substituted or unsubstituted C₃-C₈cycloalkyl, each substituted or unsubstituted heterocycloalkyl is asubstituted or unsubstituted 3 to 8 membered heterocycloalkyl, eachsubstituted or unsubstituted aryl is a substituted or unsubstitutedC₆-C₁₀ aryl, and/or each substituted or unsubstituted heteroaryl is asubstituted or unsubstituted 5 to 10 membered heteroaryl. In someembodiments of the compounds herein, each substituted or unsubstitutedalkelyene (e.g., alkylene, alkenylene, or alkynylene) is a substitutedor unsubstituted C₁-C₂₀ alkylene, each substituted or unsubstitutedheteroalkelyene is a substituted or unsubstituted 2 to 20 memberedheteroalkylene, each substituted or unsubstituted cycloalkelyene is asubstituted or unsubstituted C₃-C₈ cycloalkylene, each substituted orunsubstituted heterocycloalkelyene is a substituted or unsubstituted 3to 8 membered heterocycloalkylene, each substituted or unsubstitutedarylene is a substituted or unsubstituted C₆-C₁₀ arylene, and/or eachsubstituted or unsubstituted heteroarylene is a substituted orunsubstituted 5 to 10 membered heteroarylene.

In some embodiments, each substituted or unsubstituted alkyl is asubstituted or unsubstituted C₁-C₈ alkyl, each substituted orunsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8membered heteroalkyl, each substituted or unsubstituted cycloalkyl is asubstituted or unsubstituted C₃-C₇ cycloalkyl, each substituted orunsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7membered heterocycloalkyl, each substituted or unsubstituted aryl is asubstituted or unsubstituted C₆-C₁₀ aryl, and/or each substituted orunsubstituted heteroaryl is a substituted or unsubstituted 5 to 9membered heteroaryl. In some embodiments, each substituted orunsubstituted alkelyene (e.g., alkylene, alkenylene, or alkynylene) is asubstituted or unsubstituted C₁-C₈ alkylene, each substituted orunsubstituted heteroalkelyene is a substituted or unsubstituted 2 to 8membered heteroalkylene, each substituted or unsubstitutedcycloalkelyene is a substituted or unsubstituted C₃-C₇ cycloalkylene,each substituted or unsubstituted heterocycloalkelyene is a substitutedor unsubstituted 3 to 7 membered heterocycloalkylene, each substitutedor unsubstituted arylene is a substituted or unsubstituted C₆-C₁₀arylene, and/or each substituted or unsubstituted heteroarylene is asubstituted or unsubstituted 5 to 9 membered heteroarylene. In someembodiments, the compound is a chemical species set forth in theExamples section, figures, or tables below.

Certain compounds of the present invention possess asymmetric carbonatoms (optical or chiral centers) or double bonds; the enantiomers,racemates, diastereomers, tautomers, geometric isomers, stereoisometricforms that may be defined, in terms of absolute stereochemistry, as (R)—or (S)— or, as (D)- or (L)- for amino acids, and individual isomers areencompassed within the scope of the present invention. The compounds ofthe present invention do not include those that are known in art to betoo unstable to synthesize and/or isolate. The present invention ismeant to include compounds in racemic and optically pure forms.Optically active (R)— and (S)—, or (D)- and (L)-isomers may be preparedusing chiral synthons or chiral reagents, or resolved using conventionaltechniques. When the compounds described herein contain olefinic bondsor other centers of geometric asymmetry, and unless specified otherwise,it is intended that the compounds include both E and Z geometricisomers.

As used herein, the term “isomers” refers to compounds having the samenumber and kind of atoms, and hence the same molecular weight, butdiffering in respect to the structural arrangement or configuration ofthe atoms.

The term “tautomer,” as used herein, refers to one of two or morestructural isomers which exist in equilibrium and which are readilyconverted from one isomeric form to another.

It will be apparent to one skilled in the art that certain compounds ofthis invention may exist in tautomeric forms, all such tautomeric formsof the compounds being within the scope of the invention.

Unless otherwise stated, structures depicted herein are also meant toinclude all stereochemical forms of the structure; i.e., the R and Sconfigurations for each asymmetric center. Therefore, singlestereochemical isomers as well as enantiomeric and diastereomericmixtures of the present compounds are within the scope of the invention.

Unless otherwise stated, structures depicted herein are also meant toinclude compounds which differ only in the presence of one or moreisotopically enriched atoms. For example, compounds having the presentstructures except for the replacement of a hydrogen by a deuterium ortritium, or the replacement of a carbon by ¹³C— or ¹⁴C-enriched carbonarc within the scope of this invention.

The compounds of the present invention may also contain unnaturalproportions of atomic isotopes at one or more of the atoms thatconstitute such compounds. For example, the compounds may beradiolabeled with radioactive isotopes, such as for example tritium(³H), iodine-125 (¹²⁵I), or carbon-14 (¹⁴C) All isotopic variations ofthe compounds of the present invention, whether radioactive or not, areencompassed within the scope of the present invention.

It should be noted that throughout the application that alternatives arewritten in Markush groups, for example, each amino acid position thatcontains more than one possible amino acid. It is specificallycontemplated that each member of the Markush group should be consideredseparately, thereby comprising another embodiment, and the Markush groupis not to be read as a single unit.

“Analog,” or “analogue” is used in accordance with its plain ordinarymeaning within Chemistry and Biology and refers to a chemical compoundthat is structurally similar to another compound (i.e., a so-called“reference” compound) but differs in composition, e.g., in thereplacement of one atom by an atom of a different element, or in thepresence of a particular functional group, or the replacement of onefunctional group by another functional group, or the absolutestereochemistry of one or more chiral centers of the reference compound.Accordingly, an analog is a compound that is similar or comparable infunction and appearance but not in structure or origin to a referencecompound.

The terms “a” or “an,” as used in herein means one or more. In addition,the phrase “substituted with a[n],” as used herein, means the specifiedgroup may be substituted with one or more of any or all of the namedsubstituents. For example, where a group, such as an alkyl or heteroarylgroup, is “substituted with an unsubstituted C₁-C₂₀ alkyl, orunsubstituted 2 to 20 membered heteroalkyl,” the group may contain oneor more unsubstituted C₁-C₂₀ alkyls, and/or one or more unsubstituted 2to 20 membered heteroalkyls.

Moreover, where a moiety is substituted with an R substituent, the groupmay be referred to as “R-substituted.” Where a moiety is R-substituted,the moiety is substituted with at least one R substituent and each Rsubstituent is optionally different. Where a particular R group ispresent in the description of a chemical genus (such as Formula (I)), aRoman alphabetic symbol may be used to distinguish each appearance ofthat particular R group. For example, where multiple RP substituents arepresent, each R¹³ substituent may be distinguished as R^(13A), R^(13B),R^(13C), R^(13D), etc., wherein each of R^(13A), R^(13B), R^(13C),R^(13D), etc. is defined within the scope of the definition of R¹³ andoptionally differently.

A “detectable agent” or “detectable compound” or “detectable label” or“detectable moiety” is a composition detectable by spectroscopic,photochemical, biochemical, immunochemical, chemical, magnetic resonanceimaging, or other physical means. For example, detectable agents include¹⁸F, ³²P, ³³P, ⁴⁵ Ti, ⁴⁷Sc, ⁵²Fe, ⁵⁹Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁷⁷As,⁸⁶Y, ⁹⁰Y, ⁸⁹Sr, ⁸⁹Zr, ⁹⁴Tc, ⁹⁴Tc, ^(99m)Tc, ⁹⁹Mo, ¹⁰⁵Pd, ¹⁰⁵Rh, ¹¹¹Ag,¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁴²Pr, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁵⁴⁻¹⁵⁸¹Gd,¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁶⁹Er, ¹⁷⁵Lu, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ¹⁹⁴Ir,¹⁹⁸Au, ¹⁹⁹Au, ²¹¹At, ²¹¹Pb, ²¹²Bi, ²¹²Pb, ²¹³Bi, ²²³Ra, ²²⁵Ac, Cr, V,Mn, Fe, Co, Ni, Cu, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm,Yb, Lu, ³²P, fluorophore (e.g. fluorescent dyes), electron-densereagents, enzymes (e.g., as commonly used in an ELISA), biotin,digoxigenin, paramagnetic molecules, paramagnetic nanoparticles,ultrasmall superparamagnetic iron oxide (“USPIO”) nanoparticles, USPIOnanoparticle aggregates, superparamagnetic iron oxide (“SPIO”)nanoparticles, SPTO nanoparticle aggregates, monochrystalline iron oxidenanoparticles, monochtystalline iron oxide, nanoparticle contrastagents, liposomes or other delivery vehicles containing Gadoliniumchelate (“Gd-chelate”) molecules, Gadolinium, radioisotopes,radionuclides (e.g. carbon-11, nitrogen-13, oxygen-15, fluorine-18,rubidium-82), fluorodeoxyglucose (e.g. fluorine-18 labeled), any gammaray emitting radionuclides, positron-emitting radionuclide, radiolabeledglucose, radiolabeled water, radiolabeled ammonia, biocolloids,microbubbles (e.g. including microbubble shells including albumin,galactose, lipid, and/or polymers; microbubble gas core including air,heavy gas(es), perfluorcarbon, nitrogen, octafluoropropane, perflexanelipid microsphere, perflutren, etc.), iodinated contrast agents (e.g.iohexol, iodixanol, ioversol, iopamidol, ioxilan, iopromide,diatrizoatc, metrizoate, ioxaglatc), barium sulfate, thorium dioxide,gold, gold nanoparticles, gold nanoparticle aggregates, fluorophores,two-photon fluorophores, or haptens and proteins or other entities whichcan be made detectable. e.g., by incorporating a radiolabel into apeptide or antibody specifically reactive with a target peptide.

Radioactive substances (e.g., radioisotopes) that may be used asdetectable, imaging and/or labeling agents in accordance with theembodiments described herein include, but are not limited to, ¹⁸F, ³²P,³³P, ⁴⁵Ti, ⁴⁷Sc, ⁵²Fe, ⁵⁹Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁷⁷As, ⁸⁶Y,⁹⁰Y, ⁸⁹Sr, ⁸⁹Zr, ⁹⁴Tc, ^(99m)Tc, ⁹⁹Mo, ¹⁰⁵Pd, ¹⁰⁵Rh, ¹¹¹Ag, ¹¹¹In, ¹²³I,¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁴²Pr, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁵⁴⁻¹⁵⁸¹Gd, ¹⁶¹Tb, ¹⁶⁶Dy,¹⁶⁶Ho, ¹⁶⁹Er, ¹⁷⁵Lu, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ¹⁹⁴Ir, ¹⁹⁸Au, ²¹¹At,²¹¹Pb, ²¹²Bi, ²¹²Pb, ²¹³Bi, ²²³Ra and ²²⁵Ac. Paramagnetic ions that maybe used as additional imaging agents in accordance with the embodimentsof the disclosure include, but are not limited to, ions of transitionand lanthanide metals (e.g. metals having atomic numbers of 21-29, 42,43, 44, or 57-71). These metals include ions of Cr, V, Mn, Fe, Co, Ni,Cu, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Lu.

Examples of detectable agents include imaging agents, includingfluorescent and luminescent substances, including, but not limited to, avariety of organic or inorganic small molecules commonly referred to as“dyes,” “labels,” or “indicators.” Examples include fluorescein,rhodamine, acridine dyes, Alexa dyes, and cyanine dyes. In embodiments,the detectable moiety is a fluorescent molecule (e.g., acridine dye,cyanine, dye, fluorine dye, oxazine dye, phenanthridine dye, orrhodamine dye). In embodiments, the detectable moiety is a fluorescentmolecule (e.g., acridine dye, cyanine, dye, fluorine dye, oxazine dye,phenanthridine dye, or rhodamine dye). In embodiments, the detectablemoiety is a fluorescein isothiocyanate moiety,tetramethylrhodamine-5-(and 6)-isothiocyanate moiety, Cy2 moiety, Cy3moiety, Cy5 moiety, Cy7 moiety, 4′,6-diamidino-2-phenylindole moiety,Hoechst 33258 moiety, Hoechst 33342 moiety, Hoechst 34580 moiety,propidium-iodide moiety, or acridine orange moiety. In embodiments, thedetectable moiety is a Indo-1, Ca saturated moiety, Indo-1 Ca2+ moiety,Cascade Blue BSA pH 7.0 moiety, Cascade Blue moiety, LysoTracker Bluemoiety, Alexa 405 moiety, LysoSensor Blue pH 5.0 moiety, LysoSensor Bluemoiety, DyLight 405 moiety, DyLight 350 moiety, BFP (Blue FluorescentProtein) moiety, Alexa 350 moiety, 7-Amino-4-methylcoumarin pH 7.0moiety, Amino Coumarin moiety, AMCA conjugate moiety, Coumarin moiety,7-Hydroxy-4-methylcoumarin moiety, 7-Hydroxy-4-methylcoumarin pH 9.0moiety, 6,8-Difluoro-7-hydroxy-4-methylcoumarin pH 9.0 moiety, Hoechst33342 moiety, Pacific Blue moiety, Hoechst 33258 moiety, Hoechst33258-DNA moiety, Pacific Blue antibody conjugate pH 8.0 moiety,PO-PRO-1 moiety, PO-PRO-1-DNA moiety, POPO-1 moiety, POPO-1-DNA moiety,DAPI-DNA moiety, DAPI moiety, Marina Blue moiety, SYTOX Blue-DNA moiety,CFP (Cyan Fluorescent Protein) moiety, eCFP (Enhanced Cyan FluorescentProtein) moiety, 1-Anilinonaphthalene-8-sulfonic acid (1,8-ANS) moiety,Indo-1, Ca free moiety, 1,8-ANS (1-Anilinonaphthalene-8-sulfonic acid)moiety, BO-PRO-1-DNA moiety, BOPRO-1 moiety, BOBO-1-DNA moiety, SYTO45-DNA moiety, evoglow-Pp 1 moiety, evoglow-Bsl moiety, evoglow-Bs2moiety, Auramine O moiety, DiO moiety, LysoSensor Green pH 5.0 moiety,Cy 2 moiety, LysoSensor Green moiety, Fura-2, high Ca moiety, Fura-2Ca2+sup>moiety, SYTO 13-DNA moiety, YO-PRO-1-DNA moiety, YOYO-1-DNAmoiety, eGFP (Enhanced Green Fluorescent Protein) moiety, LysoTrackerGreen moiety, GFP (S65T) moiety, BODIPY FL. MeOH moiety, Sapphiremoiety, BODIPY FL conjugate moiety, MitoTracker Green moiety,MitaTracker Green FM, MeOH moiety, Fluorescein 0.1 M NaOH moiety,Calcein pH 9.0 moiety, Fluorescein pH 9.0 moiety, Calcein moiety,Fura-2, no Ca moiety, Fluo-4 moiety, FDA moiety, DTAF moiety,Fluorescein moiety, CFDA moiety, FITC moiety, Alexa Fluor 488hydrazide-water moiety, DyLight 488 moiety, 5-FAM pH 9.0 moiety, Alexa488 moiety, Rhodamine 110 moiety, Rhodamine 110 pH 7.0 moiety, AcridineOrange moiety, BCECF pH 5.5 moiety, PicoGreendsDNA quantitation reagentmoiety, SYBR Green I moiety, Rhodaminen Green pH 7.0 moiety, CyQUANTGR-DNA moiety, NeuroTrace 500/525, green fluorescent Nissl stain-RNAmoiety, DansylCadaverine moiety, Fluoro-Emerald moiety, Nissl moiety,Fluorescein dextran pH 8.0 moiety, Rhodamine Green moiety,5-(and-6)-Carboxy-2′, 7′-dichlorofluorescein pH 9.0 moiety,DansylCadaverine, MeOH moiety, eYFP (Enhanced Yellow FluorescentProtein) moiety, Oregon Green 488 moiety, Fluo-3 moiety, BCECF pH 9.0moiety, SBFI-Na+ moiety, Fluo-3 Ca2+ moiety, Rhodamine 123 MeOH moiety,FlAsH moiety, Calcium Green-1 Ca2+ moiety, Magnesium Green moiety,DM-NERF pH 4.0 moiety, Calcium Green moiety, Citrinc moiety, LysoSensorYellow pH 9.0 moiety, TO-PRO-1-DNA moiety, Magnesium Green Mg2+ moiety,Sodium Green Na+moiety, TOTO-1-DNA moiety, Oregon Green 514 moiety,Oregon Green 514 antibody conjugate pH 8.0 moiety, NBD-X moiety, DM-NERFpH 7.0 moiety, NBD-X, MeOH moiety. C₁-NERF pH 6.0 moiety, Alexa 430moiety, C₁-NERF pH 2.5 moiety, Lucifer Yellow, CH moiety, LysoSensorYellow pH 3.0 moiety, 6-TET, SE pH 9.0 moiety, Eosin antibody conjugatepH 8.0 moiety, Eosin moiety, 6-Carboxyrhodamine 6G pH 7.0 moiety,6-Carboxyrhodamine 6G, hydrochloride moiety, Bodipy R6G SE moiety,BODIPY R6G MeOH moiety, 6 JOE moiety, Cascade Yellow moiety, mBananamoiety, Alexa 532 moiety, Erythrosin-5-isothiocyanate pH 9.0 moiety,6-HEX, SE pH 9.0 moiety, mOrange moiety, mHoneydew moiety, Cy 3 moiety,Rhodamine B moiety, DiI moiety, 5-TAMRA-MeOH moiety, Alexa 555 moiety,DyLight 549 moiety, BODIPY TMR-X, SE moiety, BODIPY TMR-X MeOH moiety,PO-PRO-3-DNA moiety, PO-PRO-3 moiety, Rhodamine moiety, POPO-3 moiety,Alexa 546 moiety, Calcium Orange Ca2+ moiety, TRITC moiety, CalciumOrange moiety, Rhodaminephalloidin pH 7.0 moiety, MitoTracker Orangemoiety, MitoTracker Orange MeOH moiety, Phycoerythrin moiety, MagnesiumOrange moiety, R-Phycoerythrin pH 7.5 moiety, 5-TAMRA pH 7.0 moiety,5-TAMRA moiety, Rhod-2 moiety, FM 1-43 moiety, Rhod-2 Ca2+ moiety, FM1-43 lipid moiety, LOLO-1-DNA moiety, dTomato moiety, DsRed moiety,Dapoxyl (2-aminoethyl) sulfonamide moiety, Tetramethylrhodamine dextranpH 7.0 moiety, Fluor-Ruby moiety, Resorufin moiety, Resorufin pH 9.0moiety, mTangerine moiety, LysoTracker Red moiety, Lissaminerhodaminemoiety, Cy 3.5 moiety, Rhodamine Red-X antibody conjugate pH 8.0 moiety,Sulforhodamine 101 EtOH moiety, JC-1 pH 8.2 moiety, JC-1 moiety,mStrawberry moiety, MitoTracker Red moiety, MitoTracker Red, MeOHmoiety, X-Rhod-1 Ca2+ moiety, Alexa 568 moiety, 5-ROX pH 7.0 moiety,5-ROX (5-Carboxy-X-rhodamine, triethylammonium salt) moiety,BO-PRO-3-DNA moiety, BOPRO-3 moiety, BOBO-3-DNA moiety, Ethidium Bromidemoiety, ReAsH moiety, Calcium Crimson moiety, Calcium Crimson Ca2+moiety, mRFP moiety, mCherry moiety, HcRed moiety, DyLight 594 moiety,Ethidium homodimer-1-DNA moiety, Ethidiumhomodimer moiety, PropidiumIodide moiety, SYPRO Ruby moiety, Propidium Iodide-DNA moiety, Alexa 594moiety, BODIPY TR-X, SE moiety, BODIPY TR-X, MeOH moiety, BODIPY TR-Xphallacidin pH 7.0 moiety, Alexa Fluor 610 R-phycoelythrin streptavidinpH 7.2 moiety, YO-PRO-3-DNA moiety, Di-8 ANEPPS moiety,Di-8-ANEPPS-lipid moiety, YOYO-3-DNA moiety, Nile Red-lipid moiety, NileRed moiety, DyLight 633 moiety, mPlum moiety, TO-PRO-3-DNA moiety, DDAOpH 9.0 moiety, Fura Red high Ca moiety, Allophycocyanin pH 7.5 moiety,APC (allophycocyanin) moiety, Nile Blue, EtOH moiety, TOTO-3-DNA moiety,Cy 5 moiety, BODIPY 650/665-X, MeOH moiety, Alexa Fluor 647R-phycoerythrin streptavidin pH 7.2 moiety, DyLight 649 moiety, Alexa647 moiety, Fura Red Ca2+ moiety, Atto 647 moiety, Fura Red, low Camoiety, Carboxynaphthofluorescein pH 10.0 moiety, Alexa 660 moiety, Cy5.5 moiety, Alexa 680 moiety, DyLight 680 moiety, Alexa 700 moiety, FM4-64, 2% CHAPS moiety, or FM 4-64 moiety.

In embodiments, the dectable moiety is a moiety of1,1-Diethyl-4,4-carbocyanine iodide, 1,2-Diphenylacetylene.1,4-Diphenylbutadiene, 1,4-Diphenylbutadiyne, 1,6-Diphenylhexatriene,1,6-Diphenylhexatriene, 1-anilinonaphthalene-8-sulfonic acid , 2,7-Dichlorofluorescein, 2,5-DIPHENYLOXAZOLE, 2-Di-1-ASP,2-dodecylresorufin, 2-Methylbenzoxazole, 3,3-Diethylthiadicarbocyanineiodide, 4-Dimethylamino-4-Nitrostilbene, 5(6)-Carboxyfluorescein,5(6)-Carboxynaphtofluorescein, 5(6)-Carboxytetramethylrhodamine B,5-(and-6)-carboxy-2′,7′-dichlorofluorescein,5-(and-6)-carboxy-2,7-dichlorofluorescein, 5-(N-hexadecanoyDaminoeosin,5-(N-hexadecanoyl)aminoeosin, 5-chloromethylfluorescein, 5-FAM , 5-ROX ,5-TAMRA , 5-TAMRA, 6,8-difluoro-7-hydroxy-4-methylcoumarin,6,8-difluoro-7-hydroxy-4-methylcoumarin, 6-carboxyrhodamine 6G, 6-HEX,6-JOE, 6-JOE , 6-TET, 7-aminoactinomycin D,7-Benzylamino-4-Nitrobenz-2-Oxa-1,3-Diazole, 7-Methoxycoumarin-4-AceticAcid, 8-Benzyloxy-5,7-diphenylquinoline,8-Benzyloxy-5,7-diphenylquinoline, 9,10-B is (Phenylethynyl)Anthracene,9, 10-Diphenylanthracene , 9-METHYLCARBAZOLE, (CS)2Ir(μ-Cl)₂Ir(CS)₂,AAA, Acridinc Orange, Acridinc Orange , Acridinc Yellow, AcridineYellow, Adams Apple Red 680, Adirondack Green 520, Alexa Fluor 350,Alexa Fluor 405 , Alexa Fluor 430 , Alexa Fluor 430, Alexa Fluor 480,Alexa Fluor 488 , Alexa Fluor 488 , Alexa Fluor 488 hydrazide, AlexaFluor 500, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546 , AlexaFluor 546, Alexa Fluor 555, Alexa Fluor 555 , Alexa Fluor 568, AlexaFluor 594 , Alexa Fluor 594 , Alexa Fluor 594, Alexa Fluor 610, AlexaFluor 610-R-PE, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, AlexaFluor 647, Alexa Fluor 647-R-PE, Alexa Fluor 660, Alexa Fluor 680, AlexaFluor 680-APC, Alexa Fluor 680-R-PE, Alexa Fluor 700, Alexa Fluor 750,Alexa Fluor 790 , Allophycocyanin, AmCyan 1, Aminomethylcoumarin, AmplexGold (product), Amplex Red Reagent, Amplex UltraRed, Anthracene, APC,APC-Seta-750, AsRed2, ATTO 390, ATTO 425, ATTO 430LS, ATTO 465, ATTO488, ATTO 490LS, ATTO 495, ATTO 514, ATTO 520, ATTO 532, ATTO 550, ATTO565, ATTO 590, ATTO 594, ATTO 610, ATTO 620, ATTO 633, ATTO 635, ATTO647, ATTO 647N, ATTO 655, ATTO 665, ATTO 680, ATTO 700, ATTO 725, ATTO740, ATTO Oxa12, ATTO Rho3B, ATTO Rho6G, ATTO Rho11, ATTO Rho 12, ATTORho 13, ATTO Rho 14, ATTO Rho 101, ATTO Thio12, Auramine O, Azami Green,Azami Green monomeric, B-phycoerythrin, BCECF, BCECF , Bexl, Biphenyl,Birch Yellow 580, Blue-green algae, BO-PRO-1, BO-PRO-3, BOBO-1, BOBO-3,BODIPY 630 650-X, BODIPY 650/665-X, BODIPY FL, BODIPY FL, BODIPY R6G,BODIPY TMR-X, BODIPY TR-X, BODIPY TR-X Ph 7.0, BODIPY TR-X phallacidin,BODIPY-DiMe, BODIPY-Phenyl, BODIPY-TMSCC, C₃-Indocyanine,C₃-Indocyanine, C₃-Oxacyanine, C₃-Thiacyanine Dye (EtOH), C₃-ThiacyanineDye (PrOH), C5-Indocyanine, C5-Oxacyaninc, C5-Thiacyanine,C7-Indocyanine, C7-Oxacyaninc, C545T, C-Phycocyanin, Calcein, Calceinred-orange, Calcium Crimson, Calcium Green-1, Calcium Orange, Calcofluorwhite 2MR, Carboxy SNARF-1 pH 6.0, Carboxy SNARF-1 pH 9.0,Carboxynaphthofluorescein, Cascade Blue, Cascade Yellow, Catskill Green540, CBQCA, CellMask Orange, CellTrace BODIPY TR methyl ester, CellTracecalcein violet, CellTraceTM Far Red, CellTracker Blue, CellTracker RedCMTPX, CellTracker Violet BMQC, CF405M, CF405S, CF488A, CF543, CF555,CFP , CFSE, CFTM 350, CFTM 485, Chlorophyll A, Chlorophyll B, Chromeo488, Chromeo 494, Chromeo 505, Chromeo 546, Chromeo 642, Citrine,Citrine , C1OH butoxy aza-BODIPY, C1OH Cl2 aza-BODIPY, CM-H₂DCFDA,Coumarin 1, Coumarin 6, Coumarin 6, Coumarin 30, Coumarin 314, Coumarin334, Coumarin 343, Coumarin 545T, Cresyl Violet Perchlorate, Crypt°Light CF1, CryptoLight CF₂, CryptoLight CF3, CryptoLight CF4,CryptoLight CFS, CryptoLight CF6, Crystal Violet, Cumarin153, Cy2, Cy3,Cy3, Cy3.5, Cy3B, Cy3B, Cy3Cy5 ET, Cy5, Cy5, Cy5.5, Cy7, Cyanine3 NHSester, Cyanine5 carboxylic acid, Cyanine5 NHS ester, Cyclotellameneghiniana Kiitzing, CypHer5 , CypHer5 pH 9.15, CyQUANT GR, CyTrakOrange, Dabcyl SE, DAF-FM, DAMC (Weiss), dansyl cadaverine, DansylGlycine (Dioxane), DAPI, DAPI , DAPI , DAPI , DAPI (DMSO), DAPI (H₂O),Dapoxyl (2-aminocthyl)sulfonamidc, DCI, DCM, DCM, DCM (acetonitrile),DCM (MeOH), DDAO , Deep Purple , di-8-ANEPPS, DiA,Dichlorotris(1,10-phenanthroline) ruthenium(II), DiClOH C12 aza-BODIPY,DiC10Hbutoxy aza-BODIPY, DiD, DiI, DiIC18(3), DiO, DiR, Diversa Cyan-FP, Diversa Green-FP , DM-NERF pH 4.0, DOCI, Doxorubicin, DPP pH-Probe590-7.5, DPP pH-Probe 590-9.0, DPP pH-Probe 590-11.0, DPP pH-Probe590-11.0, Dragon Green, DRAQS, DsRed , DsRed , DsRed, DsRed-Express,DsRed-Express2, DsRed-Express T1 , dTomato, DY-350XL, DY-480, DY-480XLMegaStokes, DY-485, DY-485XL MegaStokes, DY-490, DY-490XL MegaStokes,DY-500, DY-500XL MegaStokes, DY-520, DY-520XL MegaStokes, DY-547,DY-549P1, DY-549P1, DY-554, DY-555, DY-557, DY-557, DY-590, DY-590,DY-615, DY-630, DY-631, DY-633, DY-635, DY-636, DY-647, DY-649P1,DY-649P1, DY-650, DY-651, DY-656, DY-673, DY-675, DY-676, DY-680,DY-681, DY-700, DY-701, DY-730, DY-731, DY-750, DY-751, DY-776, DY-782,Dye-28, Dye-33, Dye-45, Dye-304, Dye-1041, DyLight 488, DyLight 549,DyLight 594, DyLight 633, DyLight 649, DyLight 680, E2-Crimson,E2-Orange, E2-Red/Green, EBFP , ECF, ECFP, ECL Plus, eGFP , ELF 97,Emerald, Envy Green, Eosin, Eosin Y, epicocconone, EqFP611,Erythrosin-5-isothiocyanate, Ethidium bromide, ethidium homodimer-1 ,Ethyl Eosin, Ethyl Eosin, Ethyl Nile Blue A ,Ethyl-p-Dimethylaminobenzoate, Ethyl-p-Dimethylaminobenzoate, Eu203nanoparticles , Eu (Soini) , Eu(tta)3DEADIT, EvaGreen, EVOblue-30, EYFP,FAD, FITC, FITC, FlAsH (Adams), Flash Red EX, FlAsH-CCPGCC,FlAsH-CCXXCC, Fluo-3, Fluo-4, Fluo-SF, Fluorescein, Fluorescein 0.1NaOH, Fluorescein-Dibase, fluoro-emerald, Fluorol 5G, FluoSpheres blue,FluoSpheres crimson, FluoSpheres dark red, FluoSpheres orange,FluoSpheres red, FluoSpheres yellow-green, FM4-64 in CTC, FM4-64 in SDS,FM 1-43, FM 4-64, Fort Orange 600, Fura Red, Fura Red Ca free, fura-2,Fura-2 Ca free, Gadodiamide, Gd-Dtpa-Bma, Gadodiamide, Gd-Dtpa-Bma,GelGreen™, GelRed™, H9-40, HcRedl, Hemo Red 720, HiLyte Fluor 488,HiLyte Fluor 555, HiLyte Fluor 647, HiLyte Fluor 680, HiLyte Fluor 750,HiLyte Plus 555, HiLyte Plus 647, HiLyte Plus 750, HmGFP, Hoechst 33258,Hoechst 33342, Hoechst-33258 . Hoechst-33258 , Hops Yellow 560, HPTS,HPTS, HPTS, HPTS, HPTS, indo-1, Indo-1 Ca free, Ir(Cn)₂(acac),Ir(Cs)₂(acac), IR-775 chloride, IR-806, Ir-OEP-CO-Cl, IRDye® 650 Alkyne,IRDye® 650 Azide, TRDye® 650 Carboxylate, TRDye® 650 DBCO, IRDyeg^(,)650 Maleimide, TRDye® 650 NHS Ester, IRDye® 680LT Carboxylate, IRDye®680LT Maleimide, IRDye® 680LT NHS Ester, IRDye®, 680RD Alkyne, IRDye®680RD Azide, IRDye® 680RD Carboxylate, IRDye® 680RD DBCO, IRDye® 680RDMaleimide, IRDye® 680RD NHS Ester, IRDye® 700 phosphoramidite, IRDye®700DX, IRDye® 700DX, IRDye® 700DX Carboxylate, IRDye® 700DX NHS Ester,IRDye® 750 Carboxylate, IRDye® 750 Maleimide, IRDye® 750 NHS Ester,IRDye® 800 phosphoramidite, IRDye® 800CW , IRDye® 800CW Alkyne, IRDye®800CW Azide, IRDye® 800CW Carboxylate, IRDye® 800CW DBCO, IRDye® 800CWMaleimide, IRDye® 800CW NHS Ester, IRDye® 800RS, 800RS Carboxylate,IRDyc® 800RS NHS Ester, IRDye® QC-1 Carboxylate, IRDyc® QC-1 NHS Ester,Isochrysis galbana-Parke, JC-1, JC-1, JOJO-1, Jonamac Red Evitag T2,Kaede Green, Kaede Red, kusabira orange, Lake Placid 490, LDS 751,Lissamine Rhodamine (Weiss), LOLO-1, lucifer yellow CH, Lucifer YellowCH, lucifer yellow CH, Lucifer Yellow CH Dilitium salt, Lumio Green,Lumio Red, Lumogen F Orange, Lumogen Red F300, Lumogen Red F300,LysoSensor Blue DND-192, LysoSensor Green DND-153, LysoSensor GreenDND-153, LysoSensor Yellow/Blue DND-160 pH 3, LysoSensor YellowBlueDND-160, LysoTracker Blue DND-22, LysoTracker Blue DND-22, LysoTrackerGreen DND-26, LysoTracker Red DND-99, LysoTracker Yellow HCK-123, MacounRed Evitag T2, Macrolex Fluorescence Red G, Macrolex Fluorescence YellowlOGN, Macrolex Fluorescence Yellow IOGN, Magnesium Green, MagnesiumOctaethylporphyrin Magnesium Orange, Magnesium Phthalocyanine ,Magnesium Phthalocyanine , Magnesium Tetramesitylporphyrin, MagnesiumTetraphenylporphyrin, malachite green isothiocyanate, Maple Red-Orange620, Marina Blue, mBanana , mBBr, mCherry Merocyanine 540, Methyl green,Methyl green, Methyl green, Methylene Blue, Methylene Blue , mHoneyDew,MitoTracker Deep Red 633, MitoTracker Green FM, MitoTracker OrangeCMTMRos, MitoTracker Red CMXRos, monobromobimane, Monochlorobimane ,Monoraphidium, mOrange , mOrange2, mPlum, mRaspberry, mRFP, mRFP1,mRFP1.2 (Wang) , mStrawberry (Shaner) , mTangerine (Shaner) ,N,N-Bis(2,4,6-trimethylphenyl)-3,4:9,10-perylenebis(dicarboximide),NADH, Naphthalene, Naphthalene, Naphthofluorescein, Naphthofluorescein,NBD-X, NeuroTrace 500525, Nilblau perchlorate, nile blue, Nile Blue ,Nile Blue (EtOH), nile red, Nile Red, Nile Red, Nile red, Nileblue A,NIR1, NIR2, NIRS, NIR4, NIR820, Octaethylporphyrin, OH butoxyaza-BODIPY, OHCl2 aza-BODIPY, Orange Fluorescent Protein, Oregon Green488, Oregon Green 488 DHPE, Oregon Green 514, Oxazinl, Oxazin 750,Oxazine 1, Oxazine 170, P4-3, P-Quaterphenyl, P-Terphenyl, PA-GFP(post-activation), PA-GFP (pre-activation), Pacific Orange,Palladium(II) meso-tetraphenyl-tetrabenzoporphyrin., PdOEPK, PdTFPP,PerCP-Cy5.5, Perylene, Perylene, Perylene bisimide pH-Probe 550-5.0,Perylene bisimide pH-Probe 550-5.5, Perylene bisimide pH-Probe 550-6.5,Perylene Green pH-Probe 720-5.5, Perylene Green Tag pH-Probe 720-6.0,Perylene Orange pH-Probe 550-2.0, Perylene Orange Tag 550. Perylene RedpH-Probe 600-5.5, Perylenediimid, Perylne Green pH-Probe 740-5.5,Phenol, Phenylalanine, pHrodo, succinimidyl ester, Phthalocyanine,PicoGreen dsDNA quantitation reagent, Pinacyanol-Iodide, Piroxicam,Platinum(II) tetraphenyltetrabenzoporphyrin, Plum Purple , PO-PRO-1,PO-PRO-3, POPO-1, POPO-3, POPOP, Porphin, PPO, Proflavin,PromoFluor-350, PromoFluor-405 PromoFluor-415 , PromoFluor-488,PromoFluor-488 Premium , PromoFluor-488LSS PromoFluor-500LSS ,PromoFluor-505 , PromoFluor-510LSS, PromoFluor-514LSS, PromoFluor-520LSS, PromoFluor-532 , PromoFluor-546 , PromoFluor-555, PromoFluor-590,PromoFluor-610, PromoFluor-633 , PromoFluor-647, PromoFluor-670,PromoFluor-680, PromoFluor-700, PromoFluor-750, PromoFluor-770 ,PromoFluor-780 , PromoFluor-840, propidium iodide, Protoporphyrin IX,PTIR475/UF, PTIR545/UF, PtOEP, PtOEPK, PtTFPP, Pyrene, QD525, QD565,QD585, QD605, QD655, QD705, QD800, QD903, QD PbS 950, QDot 525, QDot545, QDot 565, Qdot 585, Qdot 605, Qdot 625, Qdot 655, Qdot 705, Qdot800, QpyMe2, QSY 7, QSY 7, QSY 9, QSY 21, QSY 35, quinine, QuinineSulfate , Quinine sulfate, R-phycoerythrin, R-phycoerythrin,ReAsH-CCPGCC, ReAsH-CCXXCC, Red Beads (Weiss), Redmond Red, Resorufin,resorufin, rhod-2, Rhodamin 700 perchlorate, rhodamine, Rhodamine 6G,Rhodamine 6G, Rhodamine 101, rhodamine 110, Rhodamine 123, rhodamine123, Rhodamine B, Rhodamine B, Rhodamine Green, Rhodamine pH-Probe585-7.0, Rhodamine pH-Probe 585-7.5, Rhodamine phalloidin, RhodamineRed-X, Rhodamine Red-X, Rhodamine Tag pH-Probe 585-7.0, Rhodol Green,Riboflavin, Rose Bengal, Sapphire, SBFI, SBFI Zero Na, Scenedesmus sp.,SensiLight PBXL-1, SensiLight PBXL-3, Seta 633—NHS, Seta-633—NHS,SeTau-380—NHS, SeTau-647—NHS, Snake-Eye Red 900, SNIR1, SNIR2, SNIR3,SNIR4, Sodium Green, Solophenyl flavine 7GFE 500, Spectrum Aqua,Spectrum Blue, Spectrum FRed, Spectrum Gold, Spectrum Green, SpectrumOrange, Spectrum Red, Squarylium dye III, Stains All, Stilben derivate,Stilbene, Styryl8 perchlorate, Sulfo-Cyanine3 carboxylic acid,Sulfo-Cyanine3 carboxylic acid, Sulfo-Cyanine3 NHS ester, Sulfo-Cyanine5carboxylic acid, Sulforhodamine 101, sulforhodamine 101, SulforhodamineB, Sulforhodamine G, Suncoast Yellow, SuperGlo BFP, SuperGlo GFP, SurfGreen EX, SYBR Gold nucleic acid gel stain, SYBR Green I, SYPRO Ruby,SYTO 9, SYTO 11, SYTO 13, SYTO 16, SYTO 17, SYTO 45, SYTO 59, SYTO 60,SYTO 61, SYTO 62, SYTO 82, SYTO RNASelect, SYTO RNASelect , SYTOX Blue,SYTOX Green, SYTOX Orange, SYTOX Red, T-Sapphire, Tb (Soini) , tCO,tdTomato , Tcrrylcn, Tcrrylcndiimid, testdyc, Tctra-t-Butylazaporphinc,Tetra-t-Butylnaphthalocyanine, Tetracen,Tetrakis(o-Aminophenyl)Porphyrin, Tetramesitylporphyrin,Tetramethylrhodamine, tetramethylrhodamine, Tetraphenylporphyrin,Tetraphenylporphyrin, Texas Red, Texas Red DHPE, Texas Red-X,ThiolTracker Violet, Thionin acetate, TMRE, TO-PRO-1, TO-PRO-3, Toluene,Topaz (Tsien1998), TOTO-1, TOTO-3, Tris(2,2-Bipyridyl)Ruthenium(II)chloride. , Tris(4,4-diphenyl-2,2-bipyridine) ruthenium(II) chloride.,Tris(4,7-diphenyl-1,10-phenanthroline) ruthenium(II) TMS, TRITC (Weiss),TRITC Dextran (Weiss), Tryptophan, Tyrosine, Vexl, Vybrant DyeCycleGreen stain, Vybrant DyeCycle Orange stain, Vybrant DyeCycle Violetstain, WEGFP (post-activation), WellRED D2, WellRED D3, WellRED D4,WtGFP, WtGFP (Tsien1998), X-rhod-1, Yakima Yellow, YFP, YO-PRO-1,YO-PRO-3, YOYO-1, YoYo-1 , YoYo-1 dsDNA , YoYo-1 ssDNA, YOYO-3, ZincOctaethylporphyrin, Zinc Phthalocyanine, Zinc Tetramesitylporphyrin,Zinc Tetraphenylporphyrin, ZsGreen1, or ZsYellow1.

In embodiments, the detectable label is a fluorescent dye. Inembodiments, the detectable label is a fluorescent dye capable ofexchanging energy with another fluorescent dye (e.g., fluorescenceresonance energy transfer (FRET) chromophores).

In embodiments, the detectable moiety is a moiety of a derivative of oneof the detectable moieties described immediately above, wherein thederivative differs from one of the detectable moieties immediately aboveby a modification resulting from the conjugation of the detectablemoiety to a compound described herein.

The term “cyanine” or “cyanine moiety” as described herein refers to acompound containing two nitrogen groups separated by a polymethinechain. In embodiments, the cyanine moiety has 3 methine structures (i.e.cyanine 3 or Cy3). In embodiments, the cyanine moiety has 5 methinestructures (i.e. cyanine 5 or Cy5). In embodiments, the cyanine moietyhas 7 methine structures (i.e. cyanine 7 or Cy7).

Descriptions of compounds of the present invention are limited byprinciples of chemical bonding known to those skilled in the art.Accordingly, where a group may be substituted by one or more of a numberof substituents, such substitutions are selected so as to comply withprinciples of chemical bonding and to give compounds which are notinherently unstable and/or would be known to one of ordinary skill inthe art as likely to be unstable under ambient conditions, such asaqueous, neutral, and several known physiological conditions. Forexample, a heterocycloalkyl or heteroaryl is attached to the remainderof the molecule via a ring heteroatom in compliance with principles ofchemical bonding known to those skilled in the art thereby avoidinginherently unstable compounds.

The term “pharmaceutically acceptable salts” is meant to include saltsof the active compounds that arc prepared with relatively nontoxic acidsor bases, depending on the particular substituents found on thecompounds described herein. When compounds of the present inventioncontain relatively acidic functionalities, base addition salts can beobtained by contacting the neutral form of such compounds with asufficient amount of the desired base, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable base additionsalts include sodium, potassium, calcium, ammonium, organic amino, ormagnesium salt, or a similar salt. When compounds of the presentinvention contain relatively basic functionalities, acid addition saltscan be obtained by contacting the neutral form of such compounds with asufficient amount of the desired acid, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable acid additionsalts include those derived from inorganic acids like hydrochloric,hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, maleic, malonic, benzoic, succinic,suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic,p-tolylsulfonic, citric, tartaric, oxalic, methanesulfonic, and thelike. Also included arc salts of amino acids such as arginatc and thelike, and salts of organic acids like glucuronic or galactunoric acidsand the like (see, for example, Berge et al., “Pharmaceutical Salts”,Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain specificcompounds of the present invention contain both basic and acidicfunctionalities that allow the compounds to be converted into eitherbase or acid addition salts.

Thus, the compounds of the present invention may exist as salts, such aswith pharmaceutically acceptable acids. The present invention includessuch salts. Non-limiting examples of such salts include hydrochlorides,hydrobromides, phosphates, sulfates, methanesulfonates, nitrates,maleates, acetates, citrates, fumarates, proprionates, tartrates (e.g.,(+)-tartrates, (−)-tartrates, or mixtures thereof including racemicmixtures), succinates, benzoates, and salts with amino acids such asglutamic acid, and quaternary ammonium salts (e.g. methyl iodide, ethyliodide, and the like). These salts may be prepared by methods known tothose skilled in the art.

The neutral forms of the compounds are preferably regenerated bycontacting the salt with a base or acid and isolating the parentcompound in the conventional manner. The parent form of the compound maydiffer from the various salt forms in certain physical properties, suchas solubility in polar solvents.

In addition to salt forms, the present invention provides compounds,which are in a prodrug form. Prodrugs of the compounds described hereinare those compounds that readily undergo chemical changes underphysiological conditions to provide the compounds of the presentinvention. Prodrugs of the compounds described herein may be convertedin vivo after administration. Additionally, prodrugs can be converted tothe compounds of the present invention by chemical or biochemicalmethods in an ex vivo environment, such as, for example, when contactedwith a suitable enzyme or chemical reagent.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are encompassedwithin the scope of the present invention. Certain compounds of thepresent invention may exist in multiple crystalline or amorphous forms.In general, all physical forms are equivalent for the uses contemplatedby the present invention and are intended to be within the scope of thepresent invention.

“Pharmaceutically acceptable excipient” and “pharmaceutically acceptablecarrier” refer to a substance that aids the administration of an activeagent to and absorption by a subject and can be included in thecompositions of the present invention without causing a significantadverse toxicological effect on the patient. Non-limiting examples ofpharmaceutically acceptable excipients include water, NaCl, normalsaline solutions, lactated Ringer's, normal sucrose, normal glucose,binders, fillers, disintegrants, lubricants, coatings, sweeteners,flavors, salt solutions (such as Ringer's solution), alcohols, oils,gelatins, carbohydrates such as lactose, amylose or starch, fatty acidesters, hydroxymethycellulose, polyvinyl pyrrolidine, and colors, andthe like. Such preparations can be sterilized and, if desired, mixedwith auxiliary agents such as lubricants, preservatives, stabilizers,wetting agents, emulsifiers, salts for influencing osmotic pressure,buffers, coloring, and/or aromatic substances and the like that do notdeleteriously react with the compounds of the invention. One of skill inthe art will recognize that other pharmaceutical excipients are usefulin the present invention.

The term “preparation” is intended to include the formulation of theactive compound with encapsulating material as a carrier providing acapsule in which the active component with or without other carriers, issurrounded by a carrier, which is thus in association with it.Similarly, cachets and lozenges are included. Tablets, powders,capsules, pills, cachets, and lozenges can be used as solid dosage formssuitable for oral administration.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues,wherein the polymer may optionally be conjugated to a moiety that doesnot consist of amino acids. The terms apply to amino acid polymers inwhich one or more amino acid residue is an artificial chemical mimeticof a corresponding naturally occurring amino acid, as well as tonaturally occurring amino acid polymers and non-naturally occurringamino acid polymer.

A polypeptide, or a cell is “recombinant” when it is artificial orengineered, or derived from or contains an artificial or engineeredprotein or nucleic acid (e.g. non-natural or not wild type). Forexample, a polynucleotide that is inserted into a vector or any otherheterologous location, e.g., in a genome of a recombinant organism, suchthat it is not associated with nucleotide sequences that normally flankthe polynucleotide as it is found in nature is a recombinantpolynucleotide. A protein expressed in vitro or in vivo from arecombinant polynucleotide is an example of a recombinant polypeptide.Likewise, a polynucleotide sequence that does not appear in nature, forexample a variant of a naturally occurring gene, is recombinant.

“Hybridize” shall mean the annealing of one single-stranded nucleic acid(such as a primer) to another nucleic acid based on the well-understoodprinciple of sequence complementarity. In an embodiment the othernucleic acid is a single-stranded nucleic acid. The propensity forhybridization between nucleic acids depends on the temperature and ionicstrength of their miliu, the length of the nucleic acids and the degreeof complementarity. The effect of these parameters on hybridization isdescribed in, for example, Sambrook J, Fritsch E F, Maniatis T.,Molecular cloning: a laboratory manual, Cold Spring Harbor LaboratoryPress, New York (1989). As used herein, hybridization of a primer, or ofa DNA extension product, respectively, is extendable by creation of aphosphodiester bond with an available nucleotide or nucleotide analoguecapable of forming a phosphodiester bond, therewith.

“Primer” as used herein (a primer sequence) is a short, usuallychemically synthesized oligonucleotide, of appropriate length, forexample about 18-24 bases, sufficient to hybridize to a target nucleicacid (e.g. a single stranded nucleic acid) and permit the addition of anucleotide residue thereto, or oligonucleotide or polynucleotidesynthesis therefrom, under suitable conditions well-known in the art. Inan embodiment the primer is a DNA primer, i.e. a primer consisting of,or largely consisting of, deoxyribonucleotide residues. The primers aredesigned to have a sequence that is the complement of a region oftemplate/target DNA to which the primer hybridizes. The addition of anucleotide residue to the 3′ end of a primer by formation of aphosphodiester bond results in a DNA extension product. The addition ofa nucleotide residue to the 3′ end of the DNA extension product byformation of a phosphodiester bond results in a further DNA extensionproduct. In another embodiment the primer is an RNA primer.

“Nucleoside,” as used herein, refers to a glycosyl compound consistingof a nucleobase and a 5-membered ring sugar (either ribose ordeoxyribose). Nucleosides may comprise bases such as A, C, G, T, U, oranalogues thereof. Nucleotides may be modified at the base and/or andthe sugar. in an embodiment, the nucleoside is a deoxyribonucleoside. Inanother embodiment, the nucleoside is a ribonucleoside.

“Nucleotide,” as used herein, refers to a nucleoside-5′-polyphosphatecompound, or a structural analog thereof, which can be incorporated by anucleic acid polymerase to extend a growing nucleic acid chain (such asa primer). Nucleotides may comprise bases such as A, C, G, T, U, oranalogues thereof, and may comprise 2, 3, 4, 5, 6, 7, 8, or morephosphates in the phosphate group. Nucleotides may be modified at one ormore of the base, sugar, or phosphate group. A nucleotide may have alabel or tag attached (a “labeled nucleotide” or “tagged nucleotide”).In an embodiment, the nucleotide is a deoxyribonucleotide. In anotherembodiment, the nucleotide is a ribonucleotide.

“Polymerase,” as used herein, refers to any natural or non-naturallyoccurring enzyme or other catalyst that is capable of catalyzing apolymerization reaction, such as the polymerization of nucleotidemonomers to form a nucleic acid polymer. Exemplary types of polymerasesthat may be used in the compositions and methods of the presentdisclosure include the nucleic acid polymerases such as DNA polymerase,DNA- or RNA-dependent RNA polymerase, and reverse transcriptase. In somecases, the DNA polymerase is 9° N polymerase or a variant thereof, E.Coli DNA polymerase I, Bacteriophage T4 DNA polymerase, Sequenase, TaqDNA polymerase, DNA polymerase from Bacillus stearothermophilus, Bst 2.0DNA polymerase, 9° N polymerase, 9° N polymerase (exo-)A485L/Y409V,Phi29 DNA Polymerase (y29 DNA Polymerase), T7 DNA polymerase, DNApolymerase II, DNA polymerase III holoenzyme, DNA polymerase IV, DNApolymerase V, VentR DNA polymerase, Therminator^(Tm) II DNA Polymerase,Therm inator^(Tm)III DNA Polymerase, or or Therminator™ IX DNAPolymerase..

“Solid substrate” shall mean any suitable medium present in the solidphase to which a nucleic acid or an agent may be affixed. Non-limitingexamples include chips, beads and columns.

“Contacting” is used in accordance with its plain ordinary meaning andrefers to the process of allowing at least two distinct species (e.g.chemical compounds including biomolecules or cells) to becomesufficiently proximal to react, interact or physically touch. It shouldbe appreciated; however, the resulting reaction product can be produceddirectly from a reaction between the added reagents or from anintermediate from one or more of the added reagents that can be producedin the reaction mixture.

The term “contacting” may include allowing two species to react,interact, or physically touch, wherein the two species may be a compoundas described herein and a protein or enzyme. In some embodimentscontacting includes allowing a compound described herein to interactwith a protein or enzyme that is involved in a signaling pathway.

As defined herein, the term “activation”, “activate”, “activating” andthe like in reference to a protein refers to conversion of a proteininto a biologically active derivative from an initial inactive ordeactivated state. The terms reference activation, or activating,sensitizing, or up-regulating signal transduction or enzymatic activityor the amount of a protein decreased in a disease.

The tenns “agonist,” “activator,” “upregulator,” etc. refer to asubstance capable of detectably increasing the expression or activity ofa given gene or protein. The agonist can increase expression or activity10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in comparison to acontrol in the absence of the agonist. In certain instances, expressionor activity is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold orhigher than the expression or activity in the absence of the agonist.

As defined herein, the term “inhibition”, “inhibit”, “inhibiting” andthe like in reference to a protein-inhibitor interaction meansnegatively affecting (e.g. decreasing) the activity or function of theprotein relative to the activity or function of the protein in theabsence of the inhibitor. In embodiments inhibition means negativelyaffecting (e.g. decreasing) the concentration or levels of the proteinrelative to the concentration or level of the protein in the absence ofthe inhibitor. In embodiments inhibition refers to reduction of adisease or symptoms of disease. In embodiments, inhibition refers to areduction in the activity of a particular protein target. Thus,inhibition includes, at least in part, partially or totally blockingstimulation, decreasing, preventing, or delaying activation, orinactivating, desensitizing, or down-regulating signal transduction orenzymatic activity or the amount of a protein. In embodiments,inhibition refers to a reduction of activity of a target proteinresulting from a direct interaction (e.g. an inhibitor binds to thetarget protein). In embodiments, inhibition refers to a reduction ofactivity of a target protein from an indirect interaction (e.g. aninhibitor binds to a protein that activates the target protein, therebypreventing target protein activation).

The terms “inhibitor,” “repressor” or “antagonist” or “downregulator”interchangeably refer to a substance capable of detectably decreasingthe expression or activity of a given gene or protein. The antagonistcan decrease expression or activity 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90% or more in comparison to a control in the absence of theantagonist. In certain instances, expression or activity is 1.5-fold,2-fold, 3-fold, 4-fold, 5-fold, 10-fold or lower than the expression oractivity in the absence of the antagonist.

The terms “streptavidin” and “

” refer to a tetrameric protein (including homologs, isoforms, andfunctional fragments thereof) capable of binding biotin. The termincludes any recombinant or naturally-occurring form of streptavidinvariants thereof that maintain streptavidin activity (e.g. within atleast 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% activity comparedto wildtype streptavidin).

The term “expression” includes any step involved in the production ofthe polypeptide including, but not limited to, transcription,post-transcriptional modification, translation, post-translationalmodification, and secretion. Expression can be detected usingconventional techniques for detecting protein (e.g., ELISA, Westernblotting, flow cytometry, immunofluorescence, immunohistochemistry,etc.).

An “effective amount” is an amount sufficient for a compound toaccomplish a stated purpose relative to the absence of the compound(e.g. achieve the effect for which it is administered, treat a disease,reduce enzyme activity, increase enzyme activity, reduce a signalingpathway, or reduce one or more symptoms of a disease or condition). An“activity decreasing amount,” as used herein, refers to an amount ofantagonist required to decrease the activity of an enzyme relative tothe absence of the antagonist. A “function disrupting amount,” as usedherein, refers to the amount of antagonist required to disrupt thefunction of an enzyme or protein relative to the absence of theantagonist.

A “cell” as used herein, refers to a cell carrying out metabolic orother function sufficient to preserve or replicate its genomic DNA. Acell can be identified by well-known methods in the art including, forexample, presence of an intact membrane, staining by a particular dye,ability to produce progeny or, in the case of a gamete, ability tocombine with a second gamete to produce a viable offspring. Cells mayinclude prokaryotic and eukaroytic cells. Prokaryotic cells include butare not limited to bacteria. Eukaryotic cells include but are notlimited to yeast cells and cells derived from plants and animals, forexample mammalian, insect (e.g., spodoptera) and human cells. Cells maybe useful when they are naturally nonadherent or have been treated notto adhere to surfaces, for example by trypsinization.

“Control” or “control experiment” is used in accordance with its plainordinary meaning and refers to an experiment in which the subjects orreagents of the experiment are treated as in a parallel experimentexcept for omission of a procedure, reagent, or variable of theexperiment. In some instances, the control is used as a standard ofcomparison in evaluating experimental effects. In some embodiments, acontrol is the measurement of the activity of a protein in the absenceof a compound as described herein (including embodiments and examples).

The term “modulate” is used in accordance with its plain ordinarymeaning and refers to the act of changing or varying one or moreproperties. “Modulation” refers to the process of changing or varyingone or more properties. For example, as applied to the effects of amodulator on a target protein, to modulate means to change by increasingor decreasing a property or function of the target molecule or theamount of the target molecule.

The term “aberrant” as used herein refers to different from normal. Whenused to describe enzymatic activity or protein function, aberrant refersto activity or function that is greater or less than a normal control orthe average of normal non-diseased control samples.

“Nucleic acid” or “oligonucleotide” or “polynucleotide” or grammaticalequivalents used herein means at least two nucleotides covalently linkedtogether. The term “nucleic acid” includes single-, double-, ormultiple-stranded DNA, RNA and analogs (derivatives) thereof.Oligonucleotides are typically from about 5, 6, 7, 8, 9, 10, 12, 15, 25,30, 40, 50 or more nucleotides in length, up to about 100 nucleotides inlength. Nucleic acids and polynucleotides are a polymers of any length,including longer lengths, e.g., 200, 300, 500, 1000, 2000, 3000, 5000,7000, 10,000, etc. In certain embodiments the nucleic acids hereincontain phosphodiester bonds. In other embodiments, nucleic acid analogsare included that may have alternate backbones, comprising, e.g.,phosphoramidatc, phosphorothioatc, phosphorodithioate, orO-methylphosphoroamidite linkages (see Eckstein, Oligonucleotides andAnalogues: A Practical Approach, Oxford University Press); and peptidenucleic acid backbones and linkages. Other analog nucleic acids includethose with positive backbones; non-ionic backbones, and non-ribosebackbones, including those described in U.S. Pat. Nos. 5,235,033 and5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, CarbohydrateModifications in Antisense Research, Sanghui & Cook, eds. Nucleic acidscontaining one or more carbocyclic sugars are also included within onedefinition of nucleic acids. Modifications of the ribose-phosphatebackbone may be done for a variety of reasons, e.g., to increase thestability and half-life of such molecules in physiological environmentsor as probes on a biochip. Mixtures of naturally occurring nucleic acidsand analogs can be made; alternatively, mixtures of different nucleicacid analogs, and mixtures of naturally occurring nucleic acids andanalogs may be made. A residue of a nucleic acid, as referred to herein,is a monomer of the nucleic acid (e.g., a nucleotide).

A particular nucleic acid sequence also encompasses “splice variants.”Similarly, a particular protein encoded by a nucleic acid encompassesany protein encoded by a splice variant of that nucleic acid. “Splicevariants,” as the name suggests, arc products of alternative splicing ofa gene. After transcription, an initial nucleic acid transcript may bespliced such that different (alternate) nucleic acid splice productsencode different polypeptides. Mechanisms for the production of splicevariants vary, but include alternate splicing of exons. Alternatepolypeptides derived from the same nucleic acid by read-throughtranscription are also encompassed by this definition. Any products of asplicing reaction, including recombinant forms of the splice products,are included in this definition. An example of potassium channel splicevariants is discussed in Leicher, et al., J. Biol. Chem.273(52):35095-35101 (1998).

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are near each other, and, inthe case of a secretory leader, contiguous and in reading phase.However, enhancers do not have to be contiguous. Linking is accomplishedby ligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that arc the same(i.e., about 60% identity, preferably 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% ,94%, 95%,96%, 97%, 98%, 99% or higher identity over a specified region whencompared and aligned for maximum correspondence over a comparison windowor designated region) as measured using a BLAST or BLAST 2.0 sequencecomparison algorithms with default parameters described below, or bymanual alignment and visual inspection (see, e.g., NCBI web site or thelike). Such sequences are then said to be “substantially identical.”This definition also refers to, or may be applied to, the compliment ofa test sequence. The definition also includes sequences that havedeletions and/or additions, as well as those that have substitutions. Asdescribed below, the preferred algorithms can account for gaps and thelike. Preferably, identity exists over a region that is at least about10 amino acids or 20 nucleotides in length, or more preferably over aregion that is 10-50 amino acids or 20-50 nucleotides in length. As usedherein, percent (%) amino acid sequence identity is defined as thepercentage of amino acids in a candidate sequence that are identical tothe amino acids in a reference sequence, after aligning the sequencesand introducing gaps, if necessary, to achieve the maximum percentsequence identity. Alignment for purposes of determining percentsequence identity can be achieved in various ways that are within theskill in the art, for instance, using publicly available computersoftware such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR)software. Appropriate parameters for measuring alignment, including anyalgorithms needed to achieve maximal alignment over the full-length ofthe sequences being compared can be determined by known methods.

For sequence comparisons, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Preferably.default program parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities for the test sequences relative to thereference sequence, based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 10 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),by the search for similarity method of Pearson & Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,WI), or by manual alignment and visual inspection (see, e.g., CurrentProtocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).

As used herein, the term “bioconjugate” or “bioconjugate linker” refersto the resulting association between atoms or molecules of bioconjugatereactive groups. The association can be direct or indirect. For example,a conjugate between a first bioconjugate reactive group (e.g. —NH₂,—COOH, —N-hydroxysuccinimide, or -maleimide) and a second bioconjugatereactive group (e.g., sulflwdryl, sulfur-containing amino acid, amine,amine sidechain containing amino acid, or carboxylate) provided hereincan be direct, e.g., by covalent bond or linker (e.g. a first linker ofsecond linker), or indirect, e.g., by non-covalent bond (e.g.electrostatic interactions (e.g. ionic bond, hydrogen bond, halogenbond), van der Waals interactions (e.g. dipole-dipole, dipole-induceddipole, London dispersion), ring stacking (pi effects), hydrophobicinteractions and the like). In embodiments a bioconjugate is a clickchemistry reactant moiety when the association between atoms ormolecules of bioconjugate reactive groups is direct (e.g., covalentbond, linker).

In embodiments, bioconjugates or bioconjugate linkers are formed usingbioconjugate chemistry (i.e. the association of two bioconjugatereactive groups) including, but are not limited to nucleophilicsubstitutions (e.g., reactions of amines and alcohols with acyl halides,active esters), electrophilic substitutions (e.g., enamine reactions)and additions to carbon-carbon and carbon-heteroatom multiple bonds(e.g., Michael reaction, Diels-Alder addition). These and other usefulreactions are discussed in, for example, March, ADVANCED ORGANICCHEMISTRY, 3rd Ed., John Wiley & Sons, New York, 1985; Hermanson,BIOCONJUGATE TECHNIQUES, Academic Press, San Diego, 1996; and Feeney etal., MODIFICATION OF PROTEINS; Advances in Chemistry Series, Vol. 198,American Chemical Society, Washington, D.C., 1982. In embodiments, thefirst bioconjugate reactive group (e.g., maleimide moiety) is covalentlyattached to the second bioconjugate reactive group (e.g. a sulfhydryl).In embodiments, the first bioconjugate reactive group (e.g., haloacetylmoiety) is covalently attached to the second bioconjugate reactive group(e.g. a sulfhydryl). In embodiments, the first bioconjugate reactivegroup (e.g., pyridyl moiety) is covalently attached to the secondbioconjugate reactive group (e.g. a sulfhydryl). In embodiments, thefirst bioconjugate reactive group (e.g., —N-hydroxysuccinimide moiety)is covalently attached to the second bioconjugate reactive group (e.g.an amine). In embodiments, the first bioconjugate reactive group (e.g.,maleimide moiety) is covalently attached to the second bioconjugatereactive group (e.g. a sulfhydryl). In embodiments, the firstbioconjugate reactive group (e.g., -sulfo-N-hydroxysuccinimide moiety)is covalently attached to the second bioconjugatc reactive group (e.g.an amine).

Useful bioconjugate reactive groups used for bioconjugate chemistriesherein include, for example: (a) carboxyl groups and various derivativesthereof including, but not limited to, N-hydroxysuccinimide esters,N-hydroxybenztriazole esters, acid halides, acyl imidazoles, thioesters,p-nitrophenyl esters, alkyl, alkenyl, alkenyl and aromatic esters;

(b) hydroxyl groups which can be converted to esters, ethers, aldehydes,etc.

(c) haloalkyl groups wherein the halide can be later displaced with anucleophilic group such as, for example, an amine, a carboxylatc anion,thiol anion, carbanion, or an alkoxidc ion, thereby resulting in thecovalent attachment of a new group at the site of the halogen atom;

(d) dienophile groups which are capable of participating in Diels-Alderreactions such as, for example, maleimido or maleimide groups;

(e) aldehyde or ketone groups such that subsequent derivatization ispossible via formation of carbonyl derivatives such as, for example,imines, hydrazones, semicarbazones or oximes, or via such mechanisms asGrignard addition or alkyllithium addition;

(f) sulfonyl halide groups for subsequent reaction with amines, forexample, to form sulfonamides;

(g) thiol groups, which can be converted to disulfides, reacted withacyl halides, or bonded to metals such as gold, or react withmaleimides;

(h) amine or sulfhydryl groups (e.g., present in cysteine), which canbe, for example, acylated, alkylated or oxidized;

(i) alkenes, which can undergo, for example, cycloadditions, acylation,Michael addition, etc;

(j) epoxides, which can react with, for example, amines and hydroxylcompounds;

(k) phosphoramidites and other standard functional groups useful innucleic acid synthesis;

(l) metal silicon oxide bonding; and

(m) metal bonding to reactive phosphorus groups (e.g. phosphines) toform, for example, phosphate diester bonds.

(n) azides coupled to alkynes using copper catalyzed cycloaddition clickchemistry.

(o) biotin conjugate can react with avidin or strepavidin to form aavidin-biotin complex or streptavidin-biotin complex.

The bioconjugate reactive groups can be chosen such that they do notparticipate in, or interfere with, the chemical stability of theconjugate described herein. Alternatively, a reactive functional groupcan be protected from participating in the crosslinking reaction by thepresence of a protecting group. In embodiments, the bioconjugatccomprises a molecular entity derived from the reaction of an unsaturatedbond, such as a maleimide, and a sulfhydryl group.

The terms “monophosphate” is used in accordance with its ordinarymeaning in the arts and refers to a moiety having the formula:

The term “polyphosphate” refers to at least two phosphate groups, havingthe formula:

wherein np is an integer of 1 or greater. In embodiments, np is aninteger from 0 to 5. In embodiments, np is an integer from 0 to 2. Inembodiments, np is 2.

The term “base” as used herein refers to a divalent purine or pyrimidinecompound or a derivative therof, that may be a constituent of nucleicacid (i.e. DNA or RNA, or a derivative thereof). In embodiments, thebase is a derivative of a naturally occurring DNA or RNA base (e.g., abase analogue). In embodiments the base is a hybridizing base. Inembodiments the base hybridizes to a complementary base. In embodiments,the base is capable of forming at least one hydrogen bond with acomplementary base (e.g., adenine hydrogen bonds with thymine, adeninehydrogen bonds with uracil, guanine pairs with cytosine). Non-limitingexamples of a base includes cytosine or a derivative thereof (e.g.,cytosine analogue), guanine or a derivative thereof (e.g., guanineanalogue), adenine or a derivative thereof (e.g., adenine analogue),thymine or a derivative thereof (e.g., thymine analogue), uracil or aderivative thereof (e.g., uracil analogue), hypoxanthine or a derivativethereof (e.g,. hypoxanthine analogue), xanthine or a derivative thereof(e.g., xanthine analogue), 7-methylguanine or a derivative thereof(e.g., 7-methylguanine analogue) , deaza-adenine or a derivative thereof(e.g., deaza-adenine analogue), deaza-guanine or a derivative thereof(e.g., deaza-guanine), deaza-hypoxanthine or a derivative thereof,5,6-dihydrouracil or a derivative thereof (e.g., 5,6-dihydrouracilanalogue), 5-methylcytosine or a derivative thereof (e.g.,5-methylcytosine analogue), or 5-hydroxymethylcytosine or a derivativethereof (e.g., 5-hydroxymethylcytosine analogue) moieties. Inembodiments, the base is adenine, guanine, hypoxanthine, xanthine,theobromine, caffeine, uric acid, or isoguanine. In embodiments, thebase is

The term “non-covalent linker” is used in accordance with its ordinarymeaning and refers to a divalent moiety which includes at least twomolecules that are not covalently linked to each other but do interactwith each other via a non-covalent bond (e.g. electrostatic interactions(e.g. ionic bond, hydrogen bond, halogen bond) or van der Waalsinteractions (e.g. dipole-dipole, dipole-induced dipole, Londondispersion).

The term “anchor moiety” as used herein refers to a chemical moietycapable of interacting (e.g., covalently or non-covalently) with asecond, optionally different, chemical moiety (e.g., complementaryanchor moiety binder). In embodiments, the anchor moiety is abioconjugate reactive group capable of interacting (e.g., covalently)with a complementary bioconjugate reactive group (e.g., complementaryanchor moiety reactive group). In embodiments, an anchor moiety is aclick chemistry reactant moiety.

In embodiments, the anchor moiety (an “affinity anchor moiety”) iscapable of non-covalently interacting with a second chemical moiety(e.g., complementary affinity anchor moiety binder). Non-limitingexamples of an anchor moiety include biotin, azide, trans-cyclooctene(TCO) (Melissa L, et al. J. Am. Chem. Soc., 2008, 130, 13518-13519;Marjoke F. et al. Org. Biomol. Chem., 2013, 11, 6439-6455)and phenylboric acid (PBA) (Bergseid M, et al. BioTechniques, 2000, 29,1126-1133). In embodiments, an affinity anchor moiety (e.g., biotinmoiety) interacts non-covalently with a complementary affinity anchormoiety binder (e.g., streptavidin moiety). In embodiments, an anchormoiety (e.g., azide moiety, trans-cyclooctene (TCO) moiety, phenyl boricacid (PBA) moiety) covalently binds a complementary anchor moiety binder(e.g., dibenzocyclooctyne (DBCO) moiety (Jewett J C and Bertozzi C R J.Am. Chem. Soc., 2010, 132, 3688-3690), tetrazine (TZ) moiety,salicylhydroxamic acid (SHA) moiety).

The terms “cleavable linker” or “cleavable moiety” as used herein refersto a divalent or monovalent, respectively, moiety which is capable ofbeing separated (e.g., detached, split, disconnected, hydrolyzed, astable bond within the moiety is broken) into distinct entities. Acleavable linker is cleavable (e.g., specifically cleavable) in responseto external stimuli (e.g., enzymes, nucleophilic/basic reagents,reducing agents, photo-irradiation, electrophilic/acidic reagents,organometallic and metal reagents, or oxidizing reagents). A chemicallycleavable linker refers to a linker which is capable of being split inresponse to the presence of a chemical (e.g., acid, base, oxidizingagent, reducing agent, Pd(0), tris-(2-carboxyethyl)phosphine, dilutenitrous acid, fluoride, tris(3-hydroxypropyl)phosphine), sodiumdithionite (Na₂S₂O₄), hydrazine (N₂H₄)). A chemically cleavable linkeris non-enzymatically cleavable. In embodiments, the cleavable linker iscleaved by contacting the cleavable linker with a cleaving agent. Inembodiments, the cleaving agent is sodium dithionite (Na₂S₂O₄), weakacid, hydrazine (N₂H₄), Pd(0), or light-irradiation (e.g., ultravioletradiation).

A photocleavable linker (e.g., including or consisting of ao-nitrobenzyl group) refers to a linker which is capable of being splitin response to photo-irradiation (e.g., ultraviolet radiation). Anacid-cleavable linker refers to a linker which is capable of being splitin response to a change in the pH (e.g., increased acidity). Abase-cleavable linker refers to a linker which is capable of being splitin response to a change in the pH (e.g., decreased acidity). Anoxidant-cleavable linker refers to a linker which is capable of beingsplit in response to the presence of an oxidizing agent. Areductant-cleavable linker refers to a linker which is capable of beingsplit in response to the presence of an reducing agent (e.g.,Tris(3-hydroxypropyl)phosphine). In embodiments, the cleavable linker isa dialkylketal linker (Binaulda S, et al. Chem. Commun., 2013, 49,2082-2102; Shenoi R A, et al. J. Am. Chem. Soc., 2012, 134,14945-14957), an azo linker (Rathod, K M, et al. Chem. Sci. Tran., 2013,2, 25-28; Leriche G, et al. Eur. J. Org. Chem., 2010, 23, 4360-64), anallyl linker, a cyanoethyl linker, a1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl linker, or anitrobenzyl linker.

The term “orthogonally cleavable linker” or “orthogonal cleavablelinker” as used herein refers to a cleavable linker that is cleaved by afirst cleaving agent (e.g., enzyme, nucleophilic/basic reagent, reducingagent, photo-irradiation, electrophilic/acidic reagent, organometallicand metal reagent, oxidizing reagent) in a mixture of two or moredifferent cleaving agents and is not cleaved by any other differentcleaving agent in the mixture of two or more cleaving agents. Forexample, two different cleavable linkers are both orthogonal cleavablelinkers when a mixture of the two different cleavable linkers arereacted with two different cleaving agents and each cleavable linker iscleaved by only one of the cleaving agents and not the other cleavingagent. In embodiments, an orthogonally is a cleavable linker thatfollowing cleavage the two separated entities (e.g., fluorescent dye,bioconjugate reactive group) do not further react and form a neworthogonally cleavable linker.

The term “orthogonal binding group” or “orthogonal binding molecule” asused herein refer to a binding group (e.g. anchor moiety orcomplementary anchor moiety binder) that is capable of binding a firstcomplementary binding group (e.g., complementary anchor moiety binder oranchor moiety) in a mixture of two or more different complementarybinding groups and is unable to bind any other different complementarybinding group in the mixture of two or more complementary bindinggroups. For example, two different binding groups are both orthogonalbinding groups when a mixture of the two different binding groups arereacted with two complementary binding groups and each binding groupbinds only one of the complementary binding groups and not the othercomplementary binding group. An example of a set of four orthogonalbinding groups and a set of orthogonal complementary binding groups arethe binding groups biotin, azide, trans-cyclooctene (TCO) and phenylboric acid (PBA), which specifically and efficiently bind or react withthe complementary binding groups streptavidin, dibenzocyclooctyne(DBCO), tetrazine (TZ) and salicylhydroxamic acid (SHA) respectively.

The term “orthogonal detectable label” or “orthogonal detectable moiety”as used herein refer to a detectable label (e.g. fluorescent dye ordetectable dye) that is capable of being detected and identified (e.g.,by use of a detection means (e.g., emission wavelength, physicalcharacteristic measurement)) in a mixture or a panel (collection ofseparate samples) of two or more different detectable labels. Forexample, two different detectable labels that are fluorescent dyes areboth orthogonal detectable labels when a panel of the two differentfluorescent dyes is subjected to a wavelength of light that is absorbedby one fluorescent dye but not the other and results in emission oflight from the fluorescent dye that absorbed the light but not the otherfluorescent dye. Orthogonal detectable labels may be separatelyidentified by different absorbance or emission intensities of theorthogonal detectable labels compared to each other and not only be theabsolute presence of absence of a signal. An example of a set of fourorthogonal detectable labels is the set of Rox-Labeled Tetrazine,Alexa488-Labeled SHA, Cy5-Labeled Streptavidin, and R6G-LabeledDibenzocyclooctyne.

The term “polymerase-compatible cleavable moiety” as used herein refersa cleavable moiety which does not interfere with the function of apolymerase (e.g., DNA polymerase, modified DNA polymerase). Methods fordetermining the function of a polymerase contemplated herein aredescribed in B. Rosenblum et al. (Nucleic Acids Res. 1997 Nov 15;25(22): 4500-4504); and Z. Zhu et al. (Nucleic Acids Res. 1994 Aug. 25;22(16): 3418-3422), which are incorporated by reference herein in theirentirety for all purposes. In embodiments the polymerase-compatiblecleavable moiety does not decrease the function of a polymerase relativeto the absence of the polymerase-compatible cleavable moiety. Inembodiments, the polymerase-compatible cleavable moiety does notnegatively affect DNA polymerase recognition. In embodiments, thepolymerase-compatible cleavable moiety does not negatively affect (e.g.,limit) the read length of the DNA polymerase. Additional examples of apolymerase-compatible cleavable moiety may be found in U.S. Pat. No.6,664,079, Ju J. et al. (2006) Proc Natl Acad Sci USA103(52):19635-19640.; Ruparel H. et al. (2005) Proc Natl Acad Sci USA102(17):5932-5937.; Wu J. et al. (2007) Proc Natl Acad Sci USA104(104):16462-16467;Guo J. et al. (2008) Proc Natl Acad Sci USA105(27): 9145-9150 Bentley D. R. et al. (2008) Nature 456(7218):53-59;or Hutter D. et al. (2010) Nucleosides Nucleotides & Nucleic Acids29:879-895, which are incorporated herein by reference in their entiretyfor all purposes. in embodiments, a polymerase-compatible cleavablemoiety includes an azido moiety or a dithiol linking moiety. Inembodiments, the polymerase-compatible cleavable moiety is —NH₂, —CN,—CH₃, C₂-C₆ allyl (e.g., —CH₂—CH═CH₂), methoxyalkyl (e.g., —CH₂—O—CH₃),or —CH₂N₃. In embodiments, the polymerase-compatible cleavable moietyis:

The term “allyl” as described herein refers to an unsubstitutedmethylene attached to a vinyl group (i.e. —CH═CH₂), having the formula

An “allyl linker” refers to a divalent unsubstituted methylene attachedto a vinyl group, having the formula

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit (if appropriate) of thelower limit unless the context clearly dictates otherwise, between theupper and lower limit of that range, and any other stated or interveningvalue in that stated range, is encompassed within the invention. Theupper and lower limits of these smaller ranges may independently beincluded in the smaller ranges, and are also encompassed within theinvention, subject to any specifically excluded limit in the statedrange. Where the stated range includes one or both of the limits, rangesexcluding either or both of those included limits are also included inthe invention.

Certain Embodiments

II. Compounds

In an aspect is provided a compound of the formula:

B is a base. L¹ is covalent linker. L² is covalent linker. R³ is —OH,monophosphate, diphosphate, triphosphate, polyphosphate or a nucleicacid. R^(4A) is hydrogen, —CX¹ ₃, —CHX¹ ₂, —CH₂X¹, —OCX¹ ₃, —OCH₂X¹,—OCHX¹ ₂, —CN, —OH, —SH, —NH₂, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted alkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heteroalkyl, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted cycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heterocycloalkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted aryl, or substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted heteroaryl. R^(4B)is hydrogen, —CX² ₃, —CHX² ₂, —CH₂X², —OCX² ₃, —OCH₂X², —OCHX² ₂, —CN,—OH, —SH, —NH₂, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted alkyl, substituted (e.g., substituted with a substituentgroup, size-limited substitucnt group, or lower substituent group) orunsubstituted hctcroalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted cycloalkyl, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted heterocycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted aryl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroaryl. R⁵ is a detectablelabel or anchor moiety. R⁶ is hydrogen or a polymerase-compatiblecleavable moiety. R⁷ is hydrogen or —OR^(7A), wherein R^(7A) is hydrogenor the polymerase-compatible cleavable moiety. The symbols X¹ and X² areindependently halogen.

In an aspect is provided a compound of the formula:

B is a base. L³ is a cleavable linker. R³ is —OH, monophosphate,polyphosphate or a nucleic acid. R⁵ is a detectable label or anchormoiety. R⁷ is hydrogen or —OR^(7A), wherein R^(7A) is hydrogen or

R^(8A) is independently hydrogen, CH₃, —CX³ ₃, —CHX³ ₂, —CH₂X³, —OCX³ ₃,—OCH₂X³, —OCHX³ ₂, —CN, —OH, —SH, —NH₂, substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted alkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroalkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted cycloalkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheterocycloalkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted aryl, or substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted heteroaryl. R^(8B) is independently hydrogen, CH₃, —CX⁴ ₃,—CHX⁴ ₂, —CH₂X⁴, —OCX⁴ ₃, —OCH₂X⁴, —OCHX⁴ ₂, —CN, —OH, —SH, —NH₂,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted alkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheteroalkyl, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted cycloalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heterocycloalkyl, substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted aryl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroaryl. R^(8C) isindependently hydrogen, CH₃, —CX^(8C) ₃, —CHX^(8C) ₂, —CH₂X^(8C),—OCX^(8C) ₃, —OCH₂X^(8C), —OCHX^(8C) ₂, —CN, —OH, —SH, —NH₂, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted alkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted heteroalkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedcycloalkyl, substituted (e.g., substituted with a substituent group,size-limited substitucnt group, or lower substituent group) orunsubstituted heterocycloalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted aryl, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heteroaryl. The symbols X³, X⁴, and X^(8C) areindependently halogen. In embodiments, R^(8C) is independentlyunsubstituted phenyl.

In an aspect is provided a compound of the formula:

B is a base. L³ is a cleavable linker. R³ is —OH, monophosphate,polyphosphate or a nucleic acid. R⁵ is a detectable label or anchormoiety. R⁷ is hydrogen or —OR^(7A), wherein R^(7A) is hydrogen or

R^(8A) is hydrogen, CH₃, —CX³ ₃, —CHX³ ₂, —CH₂X³, —OCX³ ₃, —OCH₂X³,—OCHX³ ₂, —CN, CN, —OH, —SH, —NH₂, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted alkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heteroalkyl, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted cycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or un substituted heterocycloalkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted aryl, or substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted heteroaryl. R^(8B)is hydrogen, CH₃, —CX⁴ ₃, —CHX⁴ ₂, —CH₂X⁴, —OCX⁴ ₃, —OCH₂X⁴, —OCHX⁴ ₂,—CN, —OH, —SH, —NH₂, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted alkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted heteroalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted cycloalkyl, substituted (c.g., substituted witha substituent group, size-limited substitucnt group, or lowersubstituent group) or unsubstituted heterocycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted aryl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroaryl. R⁹ is hydrogen,CH₃, —CX⁵ ₃, —CHX⁵ ₂, —CH₂X⁵, —OCX⁵ ₃, —OCH₂X⁵, —OCHX⁵ ₂, —CN, —OH, —SH,—NH₂, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted alkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted heteroalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted cycloalkyl, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted heterocycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted aryl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroaryl. R¹⁰ is hydrogen,—CX⁶ ₃, —CHX⁶ ₂, —CH₂X⁶, —OCX⁶ ₃, —OCH₂X⁶, —OCHX⁶ ₂, —CN, —OH, —SH,—NH₂, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted alkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted heteroalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted cycloalkyl, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted heterocycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted aryl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroaryl. R¹¹ is hydrogen,CH₃, —CX⁷ ₃, —CHX⁷ ₂, —CH₂X⁷, —OCX⁷ ₃, —OCH₂X⁷, —OCHX⁷ ₂, —CN, —OH, —SH,—NH₂, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted alkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted heteroalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted cycloalkyl, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted heterocycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted aryl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroaryl. The symbols X³,X⁴, X⁵, X⁶ and X⁷ are independently halogen.

In another aspect is provided a compound of the formula:

R^(7A) is hydrogen or a polymerase-compatible cleavable moiety; R^(8A)is independently hydrogen, CH₃, —CX³ ₃, —CHX³ ₂, —CH₂X³, —OCX³ ₃,—OCH₂X³, —OCHX³ ₂, —CN, —OH, —SH, —NH₂, substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted alkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroalkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted cycloalkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheterocycloalkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted aryl, or substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted heteroaryl. R^(8B) is independently hydrogen, CH₃, —CX⁴ ₃,—CHX⁴ ₂, —CH₂X⁴, —OCX⁴ ₃, —OCH₂X⁴, —OCHX⁴ ₂, —CN, —OH, —SH, —NH₂,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted alkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheteroalkyl, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted cycloalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heterocycloalkyl, substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted aryl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroaryl. R⁹ isindependently hydrogen, CH₃, —CX⁵ ₃, —CHX⁵ ₂, —CH₂X⁵, —OCX⁵ ₃, —OCH₂X⁵,—OCHX⁵ ₂, —CN, —OH, —SH, —NH₂, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted alkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heteroalkyl, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted cycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heterocycloalkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted aryl, or substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted heteroaryl. R¹⁰ isindependently hydrogen, —CX⁶ ₃, —CHX⁶ ₂, —CH₂X⁶, —OCX⁶ ₃, —OCH₂X⁶,—OCHX⁶ ₂, —CN, —OH, —SH, —NH₂, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted alkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heteroalkyl, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted cycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heterocycloalkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted aryl, or substituted(e.g., substituted with a substituent group, size-limited substitucntgroup, or lower substituent group) or unsubstituted heteroaryl. Rn isindependently hydrogen, CH₃, —CX⁷ ₃, —CHX¹ ₂, —CH₂X⁷, —OCX⁷ ₃, —OCH₂X¹,—OCHX⁷ ₂, —CN, —OH, —SH, —NH₂, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted alkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heteroalkyl, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted cycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heterocycloalkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted aryl, or substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted heteroaryl. Thesymbols X³, X⁴, X⁵, X⁶ and X⁷ are independently halogen. The symbol m isindependently an integer from 1 to 4.

In an aspect is provided a compound of the formula:

R^(7A) is hydrogen or a polymerase-compatible cleavable moiety; R_(8A)is independently hydrogen, CH₃, —CX³ ₃, —CHX³ ₂, —CH₂X³, —OCX³ ₃,—OCH₂X³, —OCHX³ ₂, —CN, —OH, —SH, —NH₂, substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted alkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroalkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted cycloalkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheterocycloalkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted aryl, or substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted heteroaryl. R^(8B) is independently hydrogen, CH₃, —CX⁴ ₃,—CHX⁴ ₂, —CH₂X⁴, —OCX⁴ ₃, —OCH₂X⁴, —OCHX⁴ ₂, —CN, —OH, —SH, —NH₂,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted alkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheteroalkyl, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted cycloalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heterocycloalkyl, substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted aryl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted bete roaryl R⁹ isindependently hydrogen, CH₃, —CX⁵ ₃, —CHX⁵ ₂, —CH₂X⁵, —OCX⁵ ₃, —OCH₂X⁵,—OCHX⁵ ₂, —CN, —OH, —SH, —NH₂, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted alkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heteroalkyl, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted cycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heterocycloalkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted aryl, or substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted heteroaryl. R¹⁰ isindependently hydrogen, CH₃, —CX⁶ ₃, —CHX⁶ ₂, —OCX⁶ ₃, —OCH₂X⁶, —OCHX⁶₂, —CN, —OH, —SH, —NH₂, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted alkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heteroalkyl, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted cycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heterocycloalkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted aryl, or substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted heteroaryl. R¹¹ isindependently hydrogen, CH₃, —CX⁷ ₃, —CHX¹ ₂, —CH₂X⁷, —OCX⁷ ₃, —OCH₂X¹,—OCHX⁷ ₂, —CN, —OH, —SH, —NH₂, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted alkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heteroalkyl, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted cycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heterocycloalkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted aryl, or substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted heteroaryl. Thesymbols X³, X⁴, X⁵, X⁶ and X⁷ are independently halogen.

In an aspect is provided a the formula:

R^(8A) is hydrogen, CH₃, —CX³ ₃, —CHX³ ₂, —CH₂X³, —OCX³ ₃, —OCH₂X³,—OCHX³ ₂, —OH, —SH, —NH₂, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted alkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heteroalkyl, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted cycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heterocycloalkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted aryl, or substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted heteroaryl. R^(8B)is independently hydrogen, CH₃, —CX⁴ ₃, —CHX⁴ ₂, —CH₂X⁴, —OCX⁴ ₃,—OCH₂X⁴, —OCHX⁴ ₂, —CN, —OH, —SH, —NH₂, substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted alkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroalkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted cycloalkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or un substitutedheterocycloalkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted aryl, or substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted heteroaryl. R⁹ is independently hydrogen, CH₃, —CX⁵ ₃,—CHX⁵ ₂, —CH₂X⁵, —OCX⁵ ₃, —OCH₂X⁵, —OCHX⁵ ₂, —CN, —OH, —SH, —NH₂,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted alkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheteroalkyl, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted cycloalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heterocycloalkyl, substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted aryl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroaryl. RI° isindependently hydrogen, CH₃, —CX⁶ ₃, —CHX⁶ ₂, —OCX⁶ ₃, —OCH₂X⁶, —CN,—OH, —SH, —NH₂, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted alkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted heteroalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted cycloalkyl, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted heterocycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted aryl, or substituted (e.g.,substituted with a substitucnt group, size-limited substituent group, orlower substituent group) or unsubstituted hetcroaryl. R¹¹ isindependently hydrogen, CH₃, —CX⁷ ₃, —CHX⁷ ₂, —CH7X⁷, —OCX⁷ ₃, —OCH₂X⁷,—OCHX⁷ ₂, —CN, —OH, —SH, —NH₂, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted alkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heteroalkyl, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted cycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heterocycloalkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted aryl, or substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted heteroaryl. Thesymbols X³, X⁴, X⁵, X⁶ and X⁷ are independently halogen. The symbol m isindependently an integer from 1 to 4.

In an aspect is a compound of the formula:

R^(8A) is independently hydrogen, CH₃, —CX³ ₃, —CHX³ ₂, —CH₂X³, —OCX³ ₃,—OCH₂X³, —OCHX³ ₂, —CN, —OH, —SH, —NH₂, substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted alkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroalkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted cycloalkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheterocycloalkyl, substituted (e.g., substituted with a substitucntgroup, size-limited substituent group, or lower substituent group) orunsubstituted aryl, or substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted heteroaryl. R^(8B) is independently hydrogen, CH₃, —CX⁴ ₃.—CHX⁴ ₂, —CH₂X⁴, —OCX⁴ ₃, —OCH₂X⁴, —OCHX⁴ ₂, —CN, —OH, —SH, —NH₂,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted alkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheteroalkyl, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted cycloalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heterocycloalkyl, substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted aryl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroaryl. R⁹ isindependently hydrogen, CH₃, —CX⁵ ₃, —CHX⁵ ₂, —CH₂X⁵, —OCX⁵ ₃, —OCH₂X⁵,—OCHX⁵ ₂, —CN, —OH, —SH, —NH₂, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted alkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heteroalkyl, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted cycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heterocycloalkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted aryl, or substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted heteroaryl. R¹⁰ isindependently hydrogen, CH₃, —CX⁶ ₃, —CHX⁶ ₂, —CH₂X⁶, —OCX⁶ ₃, —OCH₂X⁶,—OCHX⁶ ₂, —CN, —OH, —SH, —NH₂, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted alkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heteroalkyl, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted cycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heterocycloalkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted aryl, or substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted heteroaryl. isindependently hydrogen, CH₃, —CX⁷ ₃, —CHX⁷ ₂, —CH₂X⁷, —OCX⁷ ₃, —OCH₂X⁷,—OCHX⁷ ₂, —CN, —OH, —SH, —NH₂, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted alkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heteroalkyl, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted cycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heterocycloalkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted aryl, or substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted heteroaryl. Thesymbols X³, X⁴, X⁵, X⁶ and X² are independently halogen.

In another aspect is provided a composition of the formula:

The symbol “———” is a non-covalent bond. B is a base. is covalentlinker. L² is covalent linker. L⁴ is a covalent linker. R³ is —OH,monophosphate, polyphosphate or a nucleic acid. is hydrogen, CH₃, —CX¹₃, —CHX¹ ₂, —CH₂X¹, —OCX¹ ₃, —OCH₂X¹, —OCHX¹ ₂, —CN, —OH, —SH, —NH₂,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted alkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheteroalkyl, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted cycloalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heterocycloalkyl, substituted (e.g., substitutedwith a substituent group, size-limited substitucnt group, or lowersubstituent group) or unsubstituted aryl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroaryl. R^(4B) ishydrogen. CH₃, —CX² ₃, —CHX² ₂, —CH₂X², —OCX² ₃, —OCH₂X², —OCHX² ₂, —CN,—OH, —SH, —NH₂, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted alkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted heteroalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted cycloalkyl, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted heterocycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted aryl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroaryl. R⁵ is an affinityanchor moiety. R⁶ is hydrogen or a polymerase-compatible cleavablemoiety. R⁷ is hydrogen or —OR^(7A), wherein R^(7A) is hydrogen or apolymerase-compatible cleavable moiety. R¹² is a complementary affinityanchor moiety binder. R¹³ is a detectable label. The symbols X¹ and X²are independently halogen.

In embodiments, b is a divalent cytosine or a derivative thereof,divalent guanine or a derivative thereof, divalent adenine or aderivative thereof, divalent thymine or a derivative thereof, divalenturacil or a derivative thereof, divalent hypoxanthine or a derivativethereof, divalent xanthine or a derivative thereof, deaza-adenine or aderivative thereof, deaza-guanine or a derivative thereof,deaza-hypoxanthine or a derivative thereof divalent 7-methylguanine or aderivative thereof, divalent 5,6-dihydrouracil or a derivative thereof,divalent 5-methylcytosine or a derivative thereof, or divalent5-hydroxymethylcytosine or a derivative thereof.

In embodiments, B is a divalent cytosine, divalent guanine, divalentadenine, divalent thymine, divalent uracil, divalent hypoxanthine,divalent xanthine, deaza-adenine, deaza-guanine, deaza-hypoxanthine or aderivative thereof divalent 7-methylguanine, divalent 5,6-dihydrouracil,divalent 5-methylcytosine, or divalent 5-hydroxymethylcytosine. Inembodiments, B is a divalent cytosine. In embodiments, B is a divalentguanine. In embodiments, B is a divalent adenine. In embodiments, B is adivalent thymine. In embodiments, B is a divalent uracil. Inembodiments, B is a divalent hypoxanthine. In embodiments, B is adivalent xanthine. In embodiments, B is a deaza-adenine. In embodiments,B is a deaza-guanine. In embodiments, B is a deaza-hypoxanthine or aderivative thereof divalent 7-methylguanine. In embodiments, B is adivalent 5,6-dihydrouracil. In embodiments, B is a divalent5-methylcytosine. In embodiments, B is a divalent5-hydroxymethylcytosine.

In embodiments, B is a divalent cytosine or a derivative thereof. Inembodiments, B is a divalent guanine or a derivative thereof. Inembodiments, B is a divalent adenine or a derivative thereof. Inembodiments, B is a divalent thymine or a derivative thereof. Inembodiments, B is a divalent uracil or a derivative thereof Inembodiments, B is a divalent hypoxanthine or a derivative thereof. Inembodiments, B is a divalent xanthine or a derivative thereof. Inembodiments, B is a deaza-adenine or a derivative thereof. Inembodiments, B is a deaza-guanine or a derivative thereof Inembodiments, B is a deaza-hypoxanthine or a derivative thereof divalent7-methylguanine or a derivative thereof. In embodiments, B is a divalent5,6-dihydrouracil or a derivative thereof. in embodiments, B is adivalent 5-methylcytosine or a derivative thereof. In embodiments, B isa divalent 5-hydroxymethylcytosine or a derivative thereof.

In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, L¹ is L^(1A)-L^(1B)-L^(1C)-L^(1D)-L^(1E); and L^(1A),L^(1B), L^(1C), L^(1D) and L^(1E) are independently a bond, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted alkylene (e.g.,alkylene, alkenylene, or alkynylene), substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted heteroalkylene (e.g.,heteroalkylene, heteroalkenylene, or heteroalkynylene), substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted cycloalkylene,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheterocycloalkylene, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted arylene, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heteroarylene; wherein at least one of L^(1A),L^(1B), L^(1C), L^(1D) and L^(1E) is not a bond.

In embodiments, L¹ is L^(1A)-L^(1B)-L^(1C)-L^(1D)-L^(1E); and L^(1A),L^(1B), L^(1C), L^(1D) and L^(1E) arc independently a bond, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted C₁-C₈ alkylene(e.g., alkylene, alkenylene, or alkynylene), substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted 2 to 8 membered heteroalkylene(e.g., heteroalkylene, heteroalkenylene, or heteroalkynylene),substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted C₃-C₈cycloalkylene, substituted (e.g., substituted with a □□bstituent group,size-limited substituent group, or lower substituent group) orunsubstituted 3 to 8 membered heterocycloalkylene, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted C₆-C₁₀ arylene, or substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 5 to 10 memberedheteroarylene; wherein at least one of L^(1A), L^(1B), L^(1C), L^(1D)and L^(1E) is not a bond.

In embodiments, L¹ is L^(1A)-L^(1B)-L^(1C)-L^(1D)-L^(1E); and L^(1A),L^(1B), L^(1C), L^(1D) and L^(1E) are independently a bond, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted C₁-C₆ alkylene(e.g., alkylene, alkenylene, or alkynylene), substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted 2 to 6 membered heteroalkylene(e.g., heteroalkylene, heteroalkenylene, or heteroalkynylene),substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted C₃-C₆cycloalkylene, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 3 to 6 membered heterocycloalkylene, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted phenyl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted 5 to 6 membered heteroarylene;wherein at least one of L^(1A), L^(1B), L^(1C), L^(1D) and L^(1E) is nota bond.

In embodiments, L¹ is a bond, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted alkylene (e.g., alkylene, alkenylene, oralkynylene), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted heteroalkylene (e.g., heteroalkylene, heteroalkenylene, orheteroalkynylene), substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted cycloalkylene, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heterocycloalkylene, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted arylene, or substituted (c.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroarylene.

In embodiments, L¹ is a bond, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted C₁-C₈ alkylene (e g , alkylene, alkenylene, oralkynylene), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 2 to 8 membered heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene), substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted C₃-C₈ cycloalkylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 8 memberedheterocycloalkylene, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted C₆-C₁₀ arylene, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 10 membered heteroarylene.

In embodiments, L¹ is a bond, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted C₁-C₆ alkylene (e.g., alkylene, alkenylene, oralkynylene), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substitucnt group) orunsubstituted 2 to 6 membered heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene), substituted (e.g., substitutedwith a substituent group, size-limited substitucnt group, or lowersubstituent group) or unsubstituted C₃-C₆ cycloalkylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 6 memberedheterocycloalkylene, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted phenyl, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 6 membered heteroarylene.

In embodiments, L¹ is L^(1A)-L^(1B)-L^(1C)-L^(1D)-L^(1E); and L^(1A),L^(1B), L^(1C), L^(1D) and L^(1E) are independently a bond, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted alkenylene (e.g.,substituted with a substituent group, or substituted with size-limitedsubstituent group), substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted heteroalkenylene; wherein at least one of L^(1A), L^(1B),L^(1C), L^(1D) and L^(1E) is not a bond.

In embodiments, L¹ is L^(1A)-L^(1B)-L^(1C)-L^(1D)-L^(1E); and L^(1A),L^(1B), L^(1C), L^(1D) and L^(1E) are independently a bond, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted C¹-C₈ alkenylene, orsubstituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted 2 to 8membered heteroalkenylene; wherein at least one of L^(1A), L^(1B),L^(1C), L^(1D) and L^(1E) is not a bond. In embodiments, L¹ is L¹ isL^(1A)-L^(1B)-L^(1C)-L^(1D)-L^(1E); and L^(1A), L^(1B), L^(1C), L^(1D)and L^(1E) are independently are independently a bond, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted C₁-C₆ alkenylene, orsubstituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted 2 to 6membered heteroalkenylene; wherein at least one of L^(1A), L^(1B),L^(1C), L^(1D) and L^(1E) is not a bond.

In embodiments, L¹ is a bond, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted alkenylene, or substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted heteroalkenylene. In embodiments, L¹is a bond, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted C₂-C₈ alkenylene, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 3 to 8 membered heteroalkenylene. Inembodiments, L¹ is a bond, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted C₂-C₆ alkenylene, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted 3 to 6 memberedheteroalkenylene.

In embodiments, L¹ is L^(1A)-L^(1B)-L^(1C)-L^(1D)-L^(1E); and L^(1A),L^(1B), L^(1C), L^(1D) and L^(1E) are independently a bond, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted alkynylene,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheteroalkynylene: wherein at least one of L^(1A), L^(2B), L^(1C), L^(1D)and L^(1E) is not a bond.

In embodiments, L¹ is L^(1A)-L^(1B)-L^(1C)-L^(1D)-L^(1E); and L^(1A),L^(1B), L^(1C), L^(1D) and L^(1E) are independently a bond, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted C₁-C₈ alkynylene, orsubstituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted 2 to 8membered heteroalkynylene; wherein at least one of L^(1A), LIB_(,)LIC_(, L)ID and _(1-,)=lE is not a bond. In embodiments, L¹ is L¹ isL^(1A)-L^(1B)-L^(1C)-L^(1D)-L^(1E); and L^(1A), L^(1B), L^(1C), L^(1D)and L^(1E) are independently a bond, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted C₁-C₆ alkynylene, or substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 2 to 6 memberedheteroalkynylene; wherein at least one of L^(1A), L^(1B), L^(1C), L^(1D)and L^(1E) is not a bond.

In embodiments, L¹ is a bond, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted alkynylene, or substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted heteroalkynylene. In embodiments, L¹is a bond, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted C₂-C₈ alkynylene, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 3 to 8 membered heteroalkynylene. Inembodiments, L¹ is a bond, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted C₂-C₆ alkynylene, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted 3 to 6 memberedheteroalkynylene.

In embodiments, L¹ is a substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted C₁-C₆ alkelyene (e.g., alkylene (e.g., alkylene,alkenylene, or alkynylene), alkenylene, or alkynylene) or substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 2 to 6 memberedheteroalkylene (e.g., heteroalkylene, heteroalkcnylenc, orheteroalkynylene). In embodiments, L¹ is an unsubstituted alkylene(e.g., alkylene, alkenylene, or alkynylene). In embodiments, L¹ is—C≡C—CH₂—.

In embodiments, L¹ is a polymer. The term “polymer” refers to a moleculeincluding repeating subunits (e.g., polymerized monomers). For example,polymeric molecules may be based upon polyethylene glycol (PEG),tetraethylene glycol (TEG), polyvinylpyrrolidone (PVP), poly(xylene), orpoly(p-xylylene). The term “polymerizable monomer” is used in accordancewith its meaning in the art of polymer chemistry and refers to acompound that may covalently bind chemically to other monomer molecules(such as other polymerizable monomers that are the same or different) toform a polymer.

In embodiments, L² is a cleavable linker. In embodiments, L² is achemically cleavable linker. In embodiments, L² is a photocleavablelinker, an acid-cleavable linker, a base-cleavable linker, anoxidant-cleavable linker, a reductant-cicavable linker, or afluoride-cleavable linker. In embodiments, L² is a photocleavablelinker. In embodiments, L² is an acid-cleavable linker. In embodiments,L² is a base-cleavable linker. In embodiments, L² is anoxidant-cleavable linker. In embodiments, L² is a reductant-cleavablelinker. In embodiments, L² is a fluoride-cleavable linker.

In embodiments, L² includes a cleavable linker. In embodiments, L²includes a chemically cleavable linker. In embodiments, L² includes aphotocleavable linker, an acid-cleavable linker, a base-cleavablelinker, an oxidant-cleavable linker, a reductant-cleavable linker, or afluoride-cleavable linker. In embodiments, L² includes a photocleavablelinker. In embodiments, L² includes an acid-cleavable linker. Inembodiments, L² includes a base-cleavable linker. In embodiments, L²includes an oxidant-cleavable linker. In embodiments, L² includes areductant-cleavable linker. In embodiments, L² includes afluoride-cleavable linker.

In embodiments, L² is a cleavable linker including a dialkylketallinker, an azo linker, an allyl linker, a cyanoethyl linker, a1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl linker, or anitrobenzyl linker. In embodiments, L² is a cleavable linker including adialkylketal linker, In embodiments, L² is a cleavable linker includingan azo linker. In embodiments, L² is a cleavable linker including anallyl linker. In embodiments, L² is a cleavable linker including acyanoethyl linker. In embodiments, L² is a cleavable linker including a1-(4.4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl linker. Inembodiments, L² is a cleavable linker including a nitrobenzyl linker.

In embodiments, L² is L^(2A)-L^(2B)-L^(2C)-L^(2D)-L^(2E); and L^(2A),L^(2B), L^(2C), L^(2D) and L^(2E) are independently a bond, —NN—,—NHC(O)—, —C(O)NH—, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted alkylene (e.g., alkylene, alkenylene, or alkynylene),substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheteroalkylene (e.g., heteroalkylene, heteroalkenylene, orheteroalkynylene), substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted cycloalkylene, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heterocycloalkylene, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted arylene, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroarylene; wherein atleast one of L^(2A), L^(2B), L^(2C), L^(2D), and L^(2E) is not a bond.

In embodiments, L² is L^(2A)-L^(2B)-L^(2C)-L^(2D)-L^(2E); and L^(2A),L^(2B), L^(2C), L^(2D) and L^(2E) are independently a bond, —NN—,—NHC(O)—, —C(O)NH—, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted C₁-C₂₀ alkylene (e.g., alkylene, alkenylene, oralkynylene), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 2 to 20 membered heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene), substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted C₃-C₂₀ cycloalkylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 20 memberedheterocycloalkylene, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted C₆-C₂₀ arylene, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 20 membered heteroarylene; wherein at leastone of L^(2A), L^(2B), L^(2C), L^(2D), and L^(2E) is not a bond.

In embodiments, L² is L^(2A)-L^(2B)-L^(2C)-L^(2D)-L^(2E); and L^(2A),L^(2B), L^(2C), L^(2D) and L^(2E) are independently a bond, —NN—,—NHC(O)—, —C(O)NH—, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted alkylene (e.g., alkylene, alkenylene, or alkynylene),substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted 2 to 10membered heteroalkylene (e.g., heteroalkylene, heteroalkenylene, orheteroalkynylene), substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted C₃-C₈ cycloalkylene, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 3 to 8 membered heterocycloalkylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted C₆-C₁₀ arylene, orsubstituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted 5 to 10membered heteroarylene; wherein at least one of L2A,_(L)2B_(, L)2C_(, L)2D_(,) and _(L)-.-2E is not a bond.

In embodiments, L² isL2A-_(L)2B_(—L)2c_(—L)2D_(—L)2E_(; and L)2A_(, L)2B_(, L)2C_(, L)2D and_(L) . 2E arc independently a bond, —NN—, —NHC(O)—, —C(O)NH—,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted C₁-C₆alkylene (e.g., alkylene, alkenylene, or alkynylene), substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted 2 to 6 membered heteroalkylene(e.g., heteroalkylene, heteroalkenylene, or heteroalkynylene),substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted C₃-C₆cycloalkylene, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 3 to 6 membered heterocycloalkylene, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted phenyl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted 5 to 6 membered heteroarylene;wherein at least one of L2A, _(L)2B_(, L)2C_(, L)2D_(,) and _(L)-.-2E isnot a bond.

In embodiments, L² is L^(2A)-L^(2B)-L^(2C)-L^(2D)-L^(2E); and L^(2A) isa bond, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted alkylene (e.g., alkylene, alkcnylcnc, or alkynylene),substituted (c.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheteroalkylene (e.g., heteroalkylene, heteroalkenylene, orheteroalkynylene). L^(2B) is a bond, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted cycloalkylene, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heterocycloalkylene,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted arylene,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheteroarylene; L^(2C) is a bond, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted cycloalkylene, substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted heterocycloalkylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted arylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted heteroarylene;L^(2D) is a bond, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted alkylene (e.g., alkylene, alkenylene, or alkynylene),substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheteroalkylene (e.g., heteroalkylene, heteroalkenylene, orheteroalkynylene); and L^(2E) is a bond, substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted alkylene (e.g., alkylene,alkenylene, or alkynylenc), substituted (e.g., substituted with asubstitucnt group, size-limited substituent group, or lower substituentgroup) or unsubstituted heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene), substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted cycloalkylene, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heterocycloalkylene,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted arylene,or substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheteroarylene; wherein at least one of L^(2A), L^(2B), L^(2C), L^(2D),and L^(2E) is not a bond.

In embodiments, L² is a bond, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted alkylene (e.g., alkylene, alkenylene, oralkynylene), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted heteroalkylene (e.g., heteroalkylene, heteroalkenylene, orheteroalkynylene), substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted cycloalkylene, substituted (e.g., substituted with asubstituent group, size-limited substitucnt group, or lower substituentgroup) or unsubstituted heterocycloalkylene, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted arylene, or substituted (c.g.,substituted with a substitucnt group, size-limited substituent group, orlower substituent group) or unsubstituted heteroarylene. In embodiments,L² is a bond, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted C₁-C₂₀ alkylene (e.g., alkylene, alkenylene, oralkynylene), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 2 to 20 membered heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene), substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted C₃-C₂₀ cycloalkylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 20 memberedheterocycloalkylene, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted C₆-C₂₀ arylene, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 20 membered heteroarylene. In embodiments,L² is a bond, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted C₁-C₈ alkylene (e.g., alkylene, alkenylene, oralkynylene), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 2 to 8 membered heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene), substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted C₃-C₈ cycloalkylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 8 memberedheterocycloalkylene, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted C₆-C₁₀ arylene, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 10 membered heteroarylene. In embodiments,L² is a bond, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted C₁-C₆ alkylene (e.g., alkylene, alkenylene, oralkynylene), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 2 to 6 membered heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene), substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted C₃-C₆ cycloalkylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 6 memberedheterocycloalkylene, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted phenyl, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 6 membered heteroarylene.

In embodiments, L² is a substituted (e.g., substituted with asubstitucnt group, size-limited substituent group, or lower substituentgroup) or unsubstituted 4 to 10 membered heteroalkylene (e.g.,heteroalkylene, heteroalkenylene, or heteroalkynylene). In embodiments,L² is a substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 4 to 8 membered heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene). In embodiments, L² is asubstituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted 4 to 6membered heteroalkylene (e.g., heteroalkylene, heteroalkenylene, orheteroalkynylene).

In embodiments, L³ is an orthogonally cleavable linker. In embodiments,L³ is a cleavable linker. In embodiments, L³ is a chemically cleavablelinker. In embodiments, L³ is a photocleavable linker, an acid-cleavablelinker, a base-cleavable linker, an oxidant-cleavable linker, areductant-cleavable linker, or a fluoride-cleavable linker. Inembodiments, L³ is a photocleavable linker. In embodiments, L³ is anacid-cleavable linker. In embodiments, L³ is a base-cleavable linker. Inembodiments, L³ is an oxidant-cleavable linker. In embodiments, L³ is areductant-cleavable linker. In embodiments, L³ is a fluoride-cleavablelinker. In embodiments, L³ is a cleavable linker including adialkylketal linker, an azo linker, an allyl linker, a cyanoethyllinker, a 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl linker, or anitrobenzyl linker. In embodiments, L³ is a cleavable linker including adialkylketal linker. In embodiments, L³ is an azo linker. Inembodiments, L³ is an allyl linker. In embodiments, L³ is a cyanoethyllinker. In embodiments, L³ is a1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl linker, or anitrobenzyl linker.

In embodiments, L³ includes an orthogonally cleavable linker. Inembodiments, L³ includes a cleavable linker. In embodiments, L³ includesa chemically cleavable linker. In embodiments, L³ includes aphotocleavable linker, an acid-cleavable linker, a base-cleavablelinker, an oxidant-cleavable linker, a reductant-cleavable linker, or afluoride-cleavable linker. In embodiments, L³ includes a photocleavablelinker. In embodiments, L³ includes an acid-cleavable linker. Inembodiments, L³ includes a base-cleavable linker. In embodiments, L³includes an oxidant-cleavable linker. In embodiments, L³ includes areductant-cleavable linker. In embodiments, L³ includes afluoride-cleavable linker. In embodiments, L³ includes a dialkylketallinker, an azo linker, an allyl linker, a cyanoethyl linker, a1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl linker, or anitrobenzyl linker. In embodiments, L³ includes a dialkylketal linker.In embodiments, L³ includes an azo linker. In embodiments, L³ includesan allyl linker. In embodiments, L³ includes a cyanoethyl linker. Inembodiments, L³ includes a1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl linker. Inembodiments, L³ includes a nitrobenzyl linker.

In embodiments, L³ is L^(3A)-L^(3B)-L^(3C)-L^(3D)-L^(3E); and L^(3A),L^(3B), L^(3C), L^(3D) and L^(3E) are independently a bond, —NN—,—NHC(O)—, —C(O)NH—, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted alkylene (e.g., alkylene, alkenylene, or alkynylene),substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheteroalkylene (e.g., heteroalkylene, heteroalkenylene, orheteroalkynylene), substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted cycloalkylene, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower sub stituentgroup) or unsubstituted heterocycloalkylene, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted arylene, or substituted (e.g.,substituted with a sub stituent group, size-limited substituent group,or lower substituent group) or unsubstituted heteroarylene; wherein atleast one of L^(3A), L^(3B), L^(3C), L^(3D), and L^(3E) is not a bond.

In embodiments, L³ is L^(3A)-L^(3B)-L^(3C)-L^(3D)-L^(3E); and L^(3A),L^(3B), L^(3C), L^(3D) and L^(3E) are independently a bond, —NN—,—NHC(O)—, —C(O)NH—, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted C₁-C₂₀ alkylene (e.g., alkylene, alkenylene, oralkynylene), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 2 to 20 membered heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene), substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted C₃-C₂₀ cycloalkylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 20 memberedheterocycloalkylene, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted C₆-C₂₀ arylene, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 20 membered heteroarylene; wherein at leastone of L^(3A), L^(3B), L^(3C), L^(3D), and L^(3E) is not a bond.

In embodiments, L³ is L^(3A)-L^(3B)-L^(3C)-L^(3D)-L^(3E); and L^(3A),L^(3B), L^(3C), L^(3D) and L^(3E) are independently a bond, —NN—,—NHC(O)—, —C(O)NH—, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted C₁-C₁₀ alkylene (e.g., alkylene, alkenylene, oralkynylene), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 2 to 10 membered heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene), substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted C₃-C₈ cycloalkylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 8 memberedheterocycloalkylene, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted C₆-C₁₀ arylene, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 10 membered heteroarylene; wherein at leastone of L^(3A), L^(3B), L^(3C), L^(3D), and L^(3E) is not a bond.

In embodiments, L³ is L^(3A)-L^(3B)-L^(3C)-L^(3D)-L^(3E); and L^(3A),L^(3B), L^(3C), L^(3D) and L^(3E) are independently a bond, —NN—,—NHC(O)—, —C(O)NH—, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted C₁-C₆ alkylene (e.g., alkylene, alkenylene, oralkynylene), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 2 to 6 membered heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene), substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted C₃-C₆ cycloalkylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 6 memberedheterocycloalkylene, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted phenyl, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 6 membered heteroarylene; wherein at leastone of L^(3A), L^(3B), L^(3C), L^(3D), and L^(3E) is not a bond.

In embodiments, L³ is L^(3A)-L^(3B)-L^(3C)-L^(3D)-L^(3E); wherein L^(3A)is a bond, —NN—, —NHC(O)—, —C(O)NH—, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted alkylene (e.g., alkylene,alkenylene, or alkynylene), substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene); L^(3B) is a bond, —NN—,—NHC(O)—, —C(O)NH—, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted cycloalkylene, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heterocycloalkylene, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted arylene, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroarylene; L^(3C) is abond, —NN—, —NHC(O)—, —C(O)NH—, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted cycloalkylene, substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted heterocycloalkylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted arylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted heteroarylene;L^(3D) is a bond, —NN—, —NHC(O)—, —C(O)NH—, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted alkylene (e.g., alkylene,alkenylene, or al kynyl ene), substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene); and L^(3E) is a bond, —NN—,—NHC(O)—, —C(O)NH—, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted alkylene (e.g., alkylene, alkenylene, or alkynylene),substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheteroalkylene (e.g., heteroalkylene, heteroalkenylene, orheteroalkynylene), substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted cycloalkylene, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heterocycloalkylene, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted arylene, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroarylene; wherein atleast one of L^(3A), L^(3B), L^(3C), L^(3D), and L^(3E) is not a bond.

In embodiments, L³ is a bond, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted alkylene (e.g., alkylene, alkenylene, oralkynylene), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted heteroalkylene (e.g., heteroalkylene, heteroalkenylene, orheteroalkynylene), substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted cycloalkylene, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heterocycloalkylene, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted arylene, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroarylene.

In embodiments, L³ is a bond, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted C₁-C₂₀ alkylene (e.g., alkylene, alkenylene, oralkynylene), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 2 to 20 membered heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene), substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted C₃-C₂₀ cycloalkylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 20 memberedheterocycloalkylene, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted C₆-C₂₀ arylene, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 20 membered heteroarylene.

In embodiments, L³ is a bond, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted C₁-C₈ alkylene (e.g., alkylene, alkenylene, oralkynylene), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 2 to 8 membered heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene), substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted C₃-C₈ cycloalkylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 8 memberedheterocycloalkylene, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted C₆-C₁₀ arylene, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 10 membered heteroarylene.

In embodiments, L³ is a bond, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted C₁-C₆ alkylene (e.g., alkylene, alkenylene, oralkynylene), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 2 to 6 membered heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene), substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted C₃-C₆ cycloalkylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 6 memberedheterocycloalkylene, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted phenyl, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 6 membered heteroarylene.

In embodiments, L³ is a substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 3 to 10 membered heteroalkylene (e.g.,heteroalkylene, heteroalkenylene, or heteroalkynylene). In embodiments,L³ is a substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 3 to 8 membered heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene). In embodiments, L³ is asubstituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted 3 to 6membered heteroalkylene (e.g., heteroalkylene, heteroalkenylene, orheteroalkynylene).

In embodiments, L³ is

wherein Z is an integer from 0 to 20, or

wherein Z is an integer from 0 to 20.

In embodiments, L²—C(CH₃)₂CH₂NHC(O)—.

In embodiments. L² is an orthogonally cleavable linker or a non-covalentlinker. In embodiments, L² includes an orthogonally cleavable linker ora non-covalent linker. In embodiments, L² is an orthogonally cleavablelinker. In embodiments, L² is a non-covalent linker.

In embodiments, -L²-R⁵ is

and z is an integer from 0 to 10.

In embodiments, -L²-R⁵ is

In embodiments, -L²-R⁵ is

In embodiments. -L²-R⁵ is

In embodiments, -L²-R⁵ is

In embodiments. -L²-R⁵ is

In embodiments, -L²-R⁵ is

In

wherein z is an integer from 0 to 10. In embodiments, -L²-R⁵ is

In embodiments, -L²-R⁵ is

In embodiments, -L²-R⁵ is

In embodiments, -L²-R⁵ is

In embodiments, −L²−R⁵ is

In embodiments, L³ is

wherein L¹ is a bond, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted a substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted alkylene (e.g., alkylene, alkenylene, or alkynylene),substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheteroalkylene (e.g., heteroalkylene, heteroalkenylene, orheteroalkynylene), substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted cycloalkylene, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted hctcrocycloalkylcnc, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted arylene, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroarylene: L² is a bond,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted asubstituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted alkylene(e.g., alkylene, alkenylene, or alkynylene), substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroalkylene (e.g.,heteroalkylene, heteroalkenylene, or heteroalkynylene), substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted cycloalkylene,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheterocycloalkylene, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted arylene, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted heteroarylene, a cleavable linker, an orthogonallycleavable linker, non-covalent linker, or -L^(2A)-L^(2B)-L^(2C)-L^(2D)-,wherein L^(2A) is a bond, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted a substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted alkylene (e.g., alkylene, alkenylene, oralkynylene), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted heteroalkylene (e.g., heteroalkylene, heteroalkenylene, orheteroalkynylene); L^(2B) is a bond substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted cycloalkylene, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heterocycloalkylene,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted arylene,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheteroarylene; L^(2C) is a bond substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted cycloalkylene, substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted heterocycloalkylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted arylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted heteroarylene; andL^(2D) is a bond, substituted (e.g., substituted with a substituentgroup, size-limited substitucnt group, or lower substituent group) orunsubstituted a substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted alkylene (e.g., alkylene, alkenylene, or alkynylene),substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheteroalkylene (e.g., heteroalkylene, heteroalkenylene, orheteroalkynylene), wherein at least one of L^(2A), L^(2B), L^(2C),L^(2D) is not a bond; R^(4A) is hydrogen, CH₃, —CX ₃, —CHX ₂, —CH₂X ,—OCX¹ ₃, —OCH₂X , —OCHX ₂, —CN, —OH, —SH, —NH₂, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted alkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroalkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted cycloalkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheterocycloalkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted aryl, or substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted heteroaryl; R^(4B) is hydrogen, CH₃, —CX² ₃, —CHX² ₂,—CH₂X², —OCX² ₃, —OCH₂X², —OCHX² ₂, —CN, —OH, —SH, —NH₂, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted alkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted heteroalkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedcycloalkyl, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted heterocycloalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted aryl, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heteroaryl; and X¹ and X² are independentlyhalogen.

In embodiments, L³ is

wherein L¹ is covalent linker; L² is covalent linker; R^(4A) ishydrogen, CH₃, —CX¹ ₃, —CHX¹ ₂, —CH₂X¹, —OCX¹ ₃, —OCH₂X¹, —OCHX¹ ₂, —CN,—OH, —SH, —NH₂, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted alkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted heteroalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted cycloalkyl, substituted (e.g., substituted witha substitucnt group, size-limited substitucnt group, or lowersubstituent group) or unsubstituted heterocycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted aryl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroaryl; R^(4B) ishydrogen, CH₃, —CX² ₃, —CHX² ₂, —CH₂X², —OCX² ₃, —OCH₂X², —OCHX² ₂, —CN,—OH, —SH, —NH₂, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted alkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted heteroalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted cycloalkyl, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted heterocycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted aryl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroaryl; and X¹ and X² areindependently halogen

In embodiments, L⁴ is an orthogonally cleavable linker. In embodiments,L⁴ is a cleavable linker. In embodiments, L⁴ is a chemically cleavablelinker. In embodiments, L⁴ is a photocleavable linker, an acid-cleavablelinker, a base-cleavable linker, an oxidant-cleavable linker, areductant-cleavable linker, or a fluoride-cleavable linker. Inembodiments, L⁴ is a photocleavable linker. In embodiments, L⁴ is anacid-cleavable linker. In embodiments, L⁴ is a base-cleavable linker. Inembodiments, L⁴ is an oxidant-cleavable linker. In embodiments, L⁴ is areductant-cleavable linker. In embodiments, L⁴ is a fluoride-cleavablelinker. In embodiments, L⁴ is a cleavable linker including adialkylketal linker, an azo linker, an allyl linker, a cyanoethyllinker, a 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl linker, or anitrobenzyl linker. In embodiments, L⁴ is a cleavable linker including adialkylketal linker. In embodiments, L⁴ is an azo linker. Inembodiments, L⁴ is an allyl linker. In embodiments, L⁴ is a cyanoethyllinker. In embodiments, L⁴ is a1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl linker, or anitrobenzyl linker.

In embodiments, L⁴ includes an orthogonally cleavable linker. Inembodiments, L⁴ includes a cleavable linker. In embodiments, L⁴ includesa chemically cleavable linker. In embodiments, L⁴ includes aphotocleavable linker, an acid-cleavable linker, a base-cleavablelinker, an oxidant-cleavable linker, a reductant-cleavable linker, or afluoride-cleavable linker. In embodiments. L⁴ includes a photocleavablelinker. In embodiments, L⁴ includes an acid-cleavable linker. Inembodiments, L⁴ includes a base-cleavable linker. In embodiments, L⁴includes an oxidant-cleavable linker. In embodiments, L⁴ includes areductant-cleavable linker. In embodiments, L⁴ includes afluoride-cleavable linker. In embodiments, L⁴ includes a dialkylketallinker, an azo linker, an allyl linker, a cyanoethyl linker, a1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl linker, or anitrobenzyl linker. In embodiments, L⁴ includes a dialkylketal linker.In embodiments, L⁴ includes an azo linker. In embodiments, L⁴ includesan allyl linker. In embodiments, L⁴ includes a cyanoethyl linker. Inembodiments, L⁴ includes a1-(4.4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl linker. Inembodiments. L⁴ includes a nitrobenzyl linker.

In embodiments, L⁴ is L^(4A)-L^(4B)-L^(4C)-L^(4D)-L^(4E). L^(4A),L^(4B), L^(4C), L^(4D), or L^(4E) are independently a bond, —NN—,—NHC(O)—, —C(O)NH—, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted alkylene (e.g., alkylene, alkenylene, or alkynylene),substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheteroalkylene (e.g., heteroalkylene, heteroalkenylene, orheteroalkynylenc), substituted (e.g., substituted with a substitucntgroup, size-limited substituent group, or lower substituent group) orunsubstituted cycloalkylene, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heterocycloalkylene, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted arylene, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroarylene; wherein atleast one of L^(4A), L^(4B), L^(4C), L^(4D), and L^(4E) is not a bond.

In embodiments, L⁴ is L^(4A)-L^(4B)-L^(4C)-L^(4D)-L^(4E). L^(4A),L^(4B), L^(4C), L^(4D), or L^(4E) are independently a bond, —NN—,—NHC(O)—, —C(O)NH—, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted C₁-C₂₀ alkylene (e.g., alkylene, alkenylene, oralkynylene), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 2 to 20 membered heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene), substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted C₃-C₂₀ cycloalkylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 20 memberedheterocycloalkylene, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted C₆-C₂₀ arylene, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 20 membered heteroarylene; wherein at leastone of L^(4A), L^(4B), L^(4C), L^(4D), and L^(4E) is not a bond.

In embodiments, L⁴ is L^(4A)-L^(4B)-L^(4C)-L^(4D)-L^(4E). L^(4A),L^(4B), L^(4C), L^(4D), or L^(4E) are independently a bond, —NN—,—NHC(O)—, —C(O)NH—, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted alkylene (e.g., alkylene, alkenylene, or alkynylene),substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted 2 to 10membered heteroalkylene (e.g., heteroalkylene, heteroalkenylene, orheteroalkynylene), substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted C₃-C₈ cycloalkylene, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 3 to 8 membered heterocycloalkylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted C₆-C₁₀ arylene, orsubstituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted 5 to 10membered heteroarylene; wherein at least one of L^(4A), L^(4B), L^(4C),L^(4D), and L^(4E) is not a bond.

In embodiments, L⁴ is L^(4A)-L^(4B)-L^(4C)-L^(4D)-L^(4E). L^(4A),L^(4B), L^(4C), L^(4D), or L^(4E) are independently a bond, —NN—,—NHC(O)—, —C(O)NH—, substituted (e.g., substituted with a substituentgroup, size-limited substitucnt group, or lower substitucnt group) orunsubstituted C₁-C₆ alkylene (e.g., alkylene, alkenylene, oralkynylene), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 2 to 6 membered heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene), substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted C₃-C₆ cycloalkylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 6 memberedheterocycloalkylene, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted phenyl, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 6 membered heteroarylene; wherein at leastone of L^(4A), L^(4B), L^(4C), L^(4D), and L^(4E) is not a bond.

In embodiments, L⁴ is L^(4A)-L^(4B)-L^(4C)-L^(4D)-L^(4E); wherein L^(4A)is a bond, —NN—, —NHC(O)—, —C(O)NH—, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted alkylene (e.g., alkylene,alkenylene, or alkynylene), substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substitucntgroup) or unsubstituted heteroalkylene (c.g., heteroalkylenc,heteroalkenylenc, or heteroalkynylene); L^(4B) is a bond, —NN—,—NHC(O)—, —C(O)NH—, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted cycloalkylene, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heterocycloalkylene, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted arylene, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroarylene; L^(4C) is abond, —NN—, —NHC(O)—, —C(O)NH—, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted cycloalkylene, substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted heterocycloalkylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted arylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted heteroarylene;L^(4D) is a bond, —NN—, —NHC(O)—, —C(O)NH—, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted alkylene (e.g., alkylene,alkenylene, or alkynylene), substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene); and L^(4E) is a bond, —NN—,—NHC(O)—, —C(O)NH—, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted alkylene (e.g., alkylene, alkenylene, or alkynylene),substituted (e.g., substituted with a substitucnt group, size-limitedsubstitucnt group, or lower substituent group) or unsubstitutedheteroalkylene (e.g., heteroalkylene, heteroalkenylene, orheteroalkynylene), substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted cycloalkylene, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heterocycloalkylene, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted arylene, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroarylene; wherein atleast one of L^(4A), L^(4B), L^(4C), L^(4D), and L^(4E) is not a bond.

In embodiments, L⁴ is a bond, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted alkylene (e.g., alkylene, alkenylene, oralkynylene), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted heteroalkylene (e.g., heteroalkylene, heteroalkenylene, orheteroalkynylene), substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted cycloalkylene, substituted (e.g., substituted with asubstituent group, size-limited substitucnt group, or lower substituentgroup) or unsubstituted heterocycloalkylene, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted arylene, or substituted (c.g.,substituted with a substitucnt group, size-limited substituent group, orlower substituent group) or unsubstituted heteroarylene.

In embodiments, L⁴ is a bond, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted C₁-C₂₀ alkylene (e.g., alkylene, alkenylene, oralkynylene), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 2 to 20 membered heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene), substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted C₃-C₂₀ cycloalkylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 20 memberedheterocycloalkylene, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted C₆-C₂₀ arylene, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 20 membered heteroarylene.

In embodiments, L⁴ is a bond, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted C₁-C₈ alkylcnc (c.g., alkylcnc, alkenylene, oralkynylene), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 2 to 8 membered heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene), substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted C₃-C₈ cycloalkylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 8 memberedheterocycloalkylene, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted C₆-C₁₀ arylene, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 10 membered heteroarylene.

In embodiments, L⁴ is a bond, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted C₁-C₆ alkylene (e.g., alkylene, alkenylene, oralkynylene), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 2 to 6 membered heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene), substituted (e.g., substitutedwith a substituent group, size-limited substitucnt group, or lowersubstitucnt group) or unsubstituted C₃-C₆ cycloalkylcnc, substituted(c.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 6 memberedheterocycloalkylene, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted phenyl, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 6 membered heteroarylene.

In embodiments, L⁴ is a substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 3 to 10 membered heteroalkylene (e.g.,heteroalkylene, heteroalkenylene, or heteroalkynylene). In embodiments,L⁴ is a substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 3 to 8 membered heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene). In embodiments, L⁴ is asubstituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted 3 to 6membered heteroalkylene (e.g., heteroalkylene, heteroalkenylene, orheteroalkynylene).

In embodiments, L^(4z) is an orthogonally cleavable linker. Inembodiments, L^(4z) is a cleavable linker. In embodiments, L^(4z) is achemically cleavable linker. In embodiments, L^(4z) is a photocleavablelinker, an acid-cleavable linker, a base-cleavable linker, anoxidant-cleavable linker, a reductant-cleavable linker, or afluoride-cleavable linker. In embodiments, L^(4z) is a photocleavablelinker. In embodiments, L^(4z) is an acid-cleavable linker. Inembodiments, L^(4z) is a base-cleavable linker. In embodiments, L^(4z)is an oxidant-cleavable linker. In embodiments, L^(4z) is areductant-cleavable linker. In embodiments, L^(4z) is a cleavable linkerincluding a dialkylketal linker, an azo linker, an allyl linker, acyanoethyl linker, a 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyllinker, or a nitrobenzyl linker.

In embodiments, L^(4z) includes an orthogonally cleavable linker. Inembodiments, L^(4z) includes a cleavable linker. In embodiments, L^(4z)includes a chemically cleavable linker. In embodiments, L^(4z) includesa photocleavable linker, an acid-cleavable linker, a base-cleavablelinker, an oxidant-cleavable linker, a reductant-cleavable linker, or afluoride-cleavable linker. In embodiments, L^(4z) includes aphotocleavable linker. In embodiments, L^(4z) includes an acid-cleavablelinker. In embodiments, L^(4z) includes a base-cleavable linker. Inembodiments, L^(4z) includes an oxidant-cleavable linker. Inembodiments, L^(4z) includes a reductant-cleavable linker. Inembodiments, L^(4z) includes a cleavable linker including a dialkylketallinker, an azo linker, an allyl linker, a cyanoethyl linker, a1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl linker, or anitrobenzyl linker.

In embodiments, L^(4z) is L^(4zA)-L^(4zB)-L^(4zC)-L^(4zD)-L^(4zE).L^(4zA), L^(4zB), L^(4zC), L^(4zD), or L^(4zE) are independently a bond,—NN—, —NHC(O)—, —C(O)NH—, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted alkylene (e.g., alkylene, alkenylene, oralkynylene), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted heteroalkylene (e.g., heteroalkylene, heteroalkenylene, orheteroalkynylene), substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted cycloalkylene, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heterocycloalkylene, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted arylene, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroarylene; wherein atleast one of L^(4zA), L^(4zB), L^(4zC), L^(4zD), and L^(4zE) is not abond.

In embodiments, L^(4z) is L^(4zA)-L^(4zB)-L^(4zC)-L^(4zD)-L^(4zE).L^(4zA), L^(4zB), L^(4zC), L^(4zD), or L^(4zE) are independently a bond,—NN—, —NHC(O)—, —C(O)NH—, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted C₁-C₂₀ alkylene (e.g., alkylene, alkenylene, oralkynylene), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 2 to 20 membered heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene), substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted C₃-C₂₀ cycloalkylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 20 memberedhetcrocycloalkylene, substituted (c.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted C₆-C₂₀ arylene, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 20 membered heteroarylene; wherein at leastone of L^(4zA), L^(4zB), L^(4zC), L^(4zD), and L^(4zE) is not a bond.

In embodiments, L^(4z) is L^(4zA)-L^(4zB)-L^(4zC)-L^(4zD)-L^(4zE).L^(4zA), L^(4zB), L^(4zC), L^(4zD), or L^(4zE) are independently a bond,—NN—, —NHC(O)—, —C(O)NH—, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted C₁-C₁₀ alkylene (e.g., alkylene, alkenylene, oralkynylene), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 2 to 10 membered heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene), substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted C₃-C₈ cycloalkylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 8 memberedheterocycloalkylene, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted C₆-C₁₀ arylene, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 10 membered heteroarylene; wherein at leastone of L^(4zA), L^(4zB), L^(4zC), L^(4zD), and L^(4zE) is not a bond.

In embodiments, L^(4z) is L^(4zA)-L^(4zB)-L^(4zC)-L^(4zD)-L^(4zE).L^(4zA), L^(4zB), L^(4zC), L^(4zD), or L^(4zE) are independently a bond,—NN—, —NHC(O)—, —C(O)NH—, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted C₁-C₆ alkylene (e.g., alkylene, alkenylene, oralkynylene), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 2 to 6 membered heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene), substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted C₃-C₆ cycloalkylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 6 memberedheterocycloalkylene, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted phenyl, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 6 membered heteroarylene; wherein at leastone of L^(4zA), L^(4zB), L^(4zC), L^(4zD), and L^(4zE) is not a bond.

In embodiments, L^(4z) is L^(4zA)-L^(4zB)-L^(4zC)-L^(4zD)-L^(4zE);wherein L^(4zA) is a bond, —NN—, —NHC(O)—, —C(O)NH—, substituted (e.g.,substituted with a substituent group, size-limited substitucnt group, orlower substituent group) or unsubstituted alkylenc (e.g., alkylenc,alkcnylcnc, or alkynylene), substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene);L^(4z) ^(B) is a bond, —NN—,—NHC(O)—, —C(O)NH—, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted cycloalkylene, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heterocycloalkylene, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted arylene, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroarylene; L^(4zC) is abond, —NN—, —NHC(O)—, —C(O)NH—, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted cycloalkylene, substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted heterocycloalkylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted arylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted heteroarylene;L^(4zD) is a bond, —NN—, —NHC(O)—, —C(O)NH—, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted alkylene (e.g., alkylenc,alkcnylcnc, or alkynylcnc), substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or hctcroalkynylcnc); and L^(4zE) is a bond, —NN—,—NHC(O)—, —C(O)NH—, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted alkylene (e.g., alkylene, alkenylene, or alkynylene),substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheteroalkylene (e.g., heteroalkylene, heteroalkenylene, orheteroalkynylene), substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted cycloalkylene, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heterocycloalkylene, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted arylene, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroarylene; wherein atleast one of L^(4zA), L^(4zB), L^(4zC), L^(4zD), and L^(4zE) is not abond.

In embodiments, L^(4z) is a bond, substituted (e.g., substituted with asubstituent group, size-limited substitucnt group, or lower substitucntgroup) or unsubstituted alkylene (e.g., alkylene, alkenylene, oralkynylene), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted hctcroalkylcne (e.g., heteroalkylene, heteroalkenylene, orheteroalkynylene), substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted cycloalkylene, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heterocycloalkylene, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted arylene, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroarylene.

In embodiments, L^(4z) is a bond, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted C₁-C₂₀ alkylene (e.g., alkylene, alkenylene, oralkynylene), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 2 to 20 membered heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene), substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted C₃-C₂₀ cycloalkylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 20 memberedheterocycloalkylene, substituted (e.g., substituted with a substitucntgroup, size-limited substitucnt group, or lower substitucnt group) orunsubstituted C₆-C₂₀ arylene, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 20 membered heteroarylene.

In embodiments, L^(4z) is a bond, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted C₁-C₈ alkylene (e.g., alkylene, alkenylene, oralkynylene), substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 2 to 8 membered heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene), substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted C₃-C₈ cycloalkylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 8 memberedheterocycloalkylene, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted C₆-C₁₀ arylene, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 10 membered heteroarvlene.

In embodiments, L^(4z) is a bond, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted C₁-C₆ alkylene (e.g., alkylene, alkcnylenc, oralkynylene), substituted (c.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 2 to 6 membered heteroalkylene (e.g., heteroalkylene,heteroalkenylcne, or hetcroalkynylene), substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted C₃-C₆ cycloalkylene, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 6 memberedheterocycloalkylene, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted phenyl, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 6 membered heteroarylene.

In embodiments, L^(4z) is a substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 3 to 10 membered heteroalkylene (e.g.,heteroalkylene, heteroalkenylene, or heteroalkynylene). In embodiments,L^(4z) is a substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 3 to 8 membered heteroalkylene (e.g., heteroalkylene,heteroalkenylene, or heteroalkynylene). In embodiments, L^(4z) is asubstituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted 3 to 6membered heteroalkylene (e.g., heteroalkylene, heteroalkenylene, orheteroalkynylene).

In embodiments, R³ is —OH. In embodiments, R³ is a monophosphate. Inembodiments, R³ is a diphosphate. In embodiments, R³ is triphosphate. Inembodiments, R³ is a polyphosphate. In embodiments, R³ is monophosphate,diphosphate, triphosphate, tetraphosphate, pentaphosphate, orhexaphosphate. In embodiments, R³ is tetraphosphate, pentaphosphate, orhexaphosphate. In embodiments, R³ is tetraphosphate. In embodiments, R³is pentaphosphate. In embodiments, R³ is hexaphosphate.

In embodiments, R³ is a nucleic acid. In embodiments, R³ is a residue ofa nucleic acid. In embodiments, R³ is a base of 10 to 10,000 base of anucleic acid. In embodiments, R³ is a 100 to 10,000 base of a nucleicacid. In embodiments, R³ is a 1000 to 10,000 base of a nucleic acid. Inembodiments, R³ is a 10 to 8,000 base of a nucleic acid. In embodiments,R³ is a 10 to 9,000 base of a nucleic acid. In embodiments, R³ is a 10to 7,000 base of a nucleic acid. In embodiments, R³ is a 10 to 6,000base of a nucleic acid. In embodiments, R³ is a 10 to 5,000 base of anucleic acid. In embodiments, R³ is a 10 to 4,000 base of a nucleicacid. In embodiments, R³ is a 10 to 3,000 base of a nucleic acid. Inembodiments, R³ is a 10 to 2,000 base of a nucleic acid. In embodiments,R³ is a 10 to 1,000 base of a nucleic acid. In embodiments, R³ is a 10to 900 base of a nucleic acid. In embodiments, R³ is a 10 to 800 base ofa nucleic acid. In embodiments, R³ is a 10 to 700 base of a nucleicacid. In embodiments, R³ is a 10 to 600 base of a nucleic acid. Inembodiments, R³ is a 10 to 500 base of a nucleic acid. In embodiments,R³ is a 10 to 400 base of a nucleic acid. In embodiments, R³ is a 10 to300 base of a nucleic acid. In embodiments, R³ is a 10 to 200 base of anucleic acid. In cmbodimcnts, R³ is a 10 to 90 base of a nucleic acid.In embodiments, R³ is a 10 to 75 base of a nucleic acid.

In embodiments, R³ is a 5 to 25 base nucleic acid. In embodiments, R³ isa 10 to 25 base nucleic acid. In embodiments, R³ is a 10 to 20 basenucleic acid. In embodiments, le is a 10 to 15 base nucleic acid. Inembodiments, R³ is a 10 to 1000 base nucleic acid. In embodiments, R³ isa 100 to 600 base nucleic acid. In embodiments, R³ is a 10 to 500 basenucleic acid. In embodiments, R³ is a 10 to 250 base nucleic acid. Inembodiments, R³ is a 10 to 100 base nucleic acid. In embodiments, R³ isa 10 to 50 base nucleic acid.

In embodiments, R³ is a nucleobase of a nucleic acid. In embodiments, R³is a nucleotide of a nucleic acid. In embodiments, R³ is a nucleoside ofa nucleic acid. In embodiments, R³ is a base of a nucleic acid 10 to10,000 nucleotides in length. In embodiments, R³ is a base of a nucleicacid 100 to 10,000 nucleotides in length.In embodiments, R³ is a base ofa nucleic acid 1000 to 10,000 nucleotides in length. In embodiments, R³is a base of a nucleic acid 10 to 8,000 nucleotides in length. Inembodiments, R³ is a base of a nucleic acid 10 to 9,000 nucleotides inlength. In embodiments, R³ is a base of a nucleic acid 10 to 7,000nucleotides in length. In embodiments, R³ is a base of a nucleic acid 10to 6,000 nucleotides in length. In embodiments, R³ is a base of anucleic acid 10 to 5,000 nucleotides in length. In embodiments, R³ is abase of a nucleic acid 10 to 4,000 base of a nucleic acid. Inembodiments, R³ is a base of a nucleic acid 10 to 3,000 nucleotides inlength. In embodiments, R³ is a base of a nucleic acid 10 to 2,000nucleotides in length. In embodiments, R³ is a base of a nucleic acid 10to 1,000 nucleotides in length. In embodiments, R³ is a base of anucleic acid 10 to 900 nucleotides in length. In embodiments, R³ is abase of a nucleic acid 10 to 800 nucleotides in length. in embodiments,R³ is a base of a nucleic acid 10 to 700 nucleotides in length. Inembodiments, R³ is a base of a nucleic acid 10 to 600 base of a nucleicacid. In embodiments, R³ is a base of a nucleic acid 10 to 500nucleotides in length. In embodiments, R³ is a base of a nucleic acid 10to 400 nucleotides in length. In embodiments, R³ is a base of a nucleicacid 10 to 300 nucleotides in length. In embodiments, R³ is a base of anucleic acid 10 to 200 nucleotides in length. In embodiments, R³ is abase of a nucleic acid 10 to 90 nucleotides in length. In embodiments,R³ is a base of a nucleic acid 10 to 75 nucleotides in length.

In embodiments, R^(4A) is hydrogen, CH₃, —CX¹ ₃, —CHX¹ ₂, —CH₂X¹, —OCX¹₃, —OCH₂X¹, —OCHX¹ ₂, —CN, —OH, —SH, —NH₂, substituted (e.g.,substituted with a substitucnt group, size-limited substituent group, orlower substituent group) or unsubstituted alkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substitucnt group) or unsubstituted heteroalkyl, substituted(c.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted cycloalkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheterocycloalkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted aryl, or substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted heteroaryl. In embodiments, R^(4A)is hydrogen, CH₃, —CX¹₃, —CHX¹ ₂, —CH₂X¹, —OCX¹ ₃, —OCH₂X¹, —OCHX¹ ₂, —CN, —OH, —SH, —NH₂,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted C₁-C₆alkyl, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 2 to 6 membered heteroalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted C₃-C₆ cycloalkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 6 memberedheterocycloalkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted phenyl, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 6 membered heteroaryl. In embodiments,R^(4A) is hydrogen.

In embodiments, R^(4A) is hydrogen, —CH₃, —CHX¹ ₂, —CH₂X¹, —CN, -Ph,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted alkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheteroalkyl, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted cycloalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heterocycloalkyl, substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted aryl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroaryl.

In embodiments, R^(4B) is hydrogen, CH₃, —CX² ₃, —CHX² ₂, —CH₂X², —OCX²₃, —OCH₂X², —OCHX² ₂, —OH, —SH, —NH₂, substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted alkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted hctcroalkyl, substituted(c.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted cycloalkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheterocycloalkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted aryl, or substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted heteroaryl.

In embodiments, R^(4B) is hydrogen, CH₃, —CX² ₃, —CHX² ₂, —CH₂X², —OCX²₃, —OCH₂X², —OCHX² ₂, —CN, —OH, —SH, —NH₂, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted C₁-C₆ alkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 2 to 6 memberedheteroalkyl, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted C₃-C₆ cycloalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 3 to 6 membered heterocycloalkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted phenyl, orsubstituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted 5 to 6membered heteroaryl. In embodiments, R^(4B) is hydrogen.

In embodiments, R^(4B) is hydrogen, —CH₃, —CX² ₃, —CHX² ₂, —CH₂X², —CN,-Ph, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted alkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted heteroalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted cycloalkyl, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted heterocycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted aryl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroaryl.

In embodiments, R^(4A) is hydrogen, —CH₃, —CX¹ ₃, —CHX¹ ₂, —CH₂X¹, —CN,-Ph, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted alkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted heteroalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted cycloalkyl, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted heterocycloalkyl, substituted (e.g.,substituted with a substituent group, sizc-limited substituent group, orlower substituent group) or unsubstituted aryl, or substituted (e.g.,substituted with a substitucnt group, size-limited substituent group, orlower substituent group) or unsubstituted hetcroaryl.

In embodiments, R^(4A) is hydrogen, —CH₃, —CX¹ ₃, —CHX¹ ₂, —CH₂X¹, —CN,-Ph, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) C₁-C₆ alkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) 2 to 6 membered heteroalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) C₃-C₆ cycloalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) 3 to 6membered heterocycloalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) phenyl, orsubstituted (e.g., substituted with a substituent group, size-limitedsubstitucnt group, or lower substitucnt group) or unsubstituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) 5 to 6 membered heteroaryl.

In embodiments, R^(4B) is hydrogen, —CH₃, —CX² ₃, —CHX² ₂, —CH₂X², —CN,-Ph, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted alkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted heteroalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted cycloalkyl, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted heterocycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted aryl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroaryl.

In embodiments, R ^(4B) is hydrogen, —CH₃, —CX² ₃, —CHX² ₂, —CH₂X², —CN,-Ph, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted C₁-C₆ alkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 2 to 6 membered heteroalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted C₃-C₆ cycloalkyl, substituted(e.g., substituted with a substitucnt group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 6 memberedheterocycloalkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted phenyl, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 6 membered heteroaryl.

In embodiments, R⁵ is a detectable label. in embodiments, R⁵ is afluorescent dye. In embodiments, R⁵ is an anchor moiety. In embodiments,R⁵ is a click chemistry reactant moiety. In embodiments, R⁵ is atrans-cyclooctene moiety or azide moiety. In embodiments, R⁵ is anaffinity anchor moiety. In embodiments, R⁵ is a biotin moiety. Inembodiments, R⁵ is a reactant for a bioconjugate reaction that forms acovalent bond between R⁵ and a second bioconjugate reaction reactant.

In embodiments, R⁵ is a fluorescent dye. In embodiments R⁵ is a AlexaFluor® 350 moiety, Alexa Fluor® 405 moiety, Alexa Fluor® 430 moiety,Alexa Fluor® 488 moiety, Alexa Fluor® 532 moiety, Alexa Fluor® 546moiety, Alexa Fluor® 555 moiety, Alexa Fluor® 568 moiety, Alexa Fluor®594 moiety, Alexa Fluor® 610 moiety, Alexa Fluor® 633 moiety, AlexaFluor® 635 moiety, Alexa Fluor® 647 moiety, Alexa Fluor® 660 moiety,Alexa Fluor® 680 moiety, Alexa Fluor® 700 moiety, Alexa Fluor® 750moiety, or Alexa Fluor® 790 moiety. In embodiments the detectable moietyis a Alexa Fluor® 488 moiety, Rhodamine 6G (R6G) moiety, ROX ReferenceDye (ROX) moiety, or Cy5 moiety.

In embodiments R⁵ is a FAM™ moiety, TET™ moiety, JOE™ moiety, VIC®moiety, HEX™ moiety, NED™ moiety, PET® moiety, ROX™ moiety, TAMRA™moiety, TET™ moiety, Texas Red® moiety, Alexa Fluor® 488 moiety,Rhodamine 6G (R6G) moiety, ROX Reference Dye (ROX) moiety, Sulfo-Cy5, orCy5 moiety. In embodiments R⁵ is a Rhodamine 6G (R6G) moiety, ROXReference Dye (ROX) moiety, Sulfo-Cy5, or Cy5 moiety.

In embodiments R⁵ is a FAM™ moiety. In embodiments R⁵ is a TET™ moiety.In embodiments R⁵ is a JOE™ moiety. In embodiments R⁵ is a VIC® moiety.In embodiments R⁵ is a HEX™ moiety. In embodiments R⁵ is a NED™ moiety.In embodiments R⁵ is a PET® moiety. In embodiments R⁵ is a ROX™ moiety.In embodiments R⁵ is a TAMRA™ moiety. In embodiments R⁵ is a TET™moiety. In embodiments R⁵ is a Texas Red® moiety. In embodiments R⁵ isan Alexa Fluor® 488 moiety. In embodiments R⁵ is a Rhodamine 6G (R6G)moiety. In embodiments R⁵ is a ROX Reference Dye (ROX) moiety. Inembodiments R⁵ is a Sulfo-Cy5. In embodiments R⁵ is a Cy5 moiety.

In embodiments, R⁵ is a biotin moiety. In embodiments, R⁵ is a biotinmoiety and R¹² is a streptavidin moiety.

In embodiments, R⁵ is

In embodiments, R⁵ is

In embodiments, R⁵ is

In embodiments, R⁵ is

In embodiments, R⁵ is

In embodiments, R⁵ is

In embodiments, R⁵ is

In embodiments, R⁵ is

In embodiments, R⁵ is

In embodiments, R⁵ is

In embodiments, R⁵ is

In embodiments, R⁵ is

In embodiments, R⁵ is —N₃. In embodiments, R⁵ is

In embodiments, R⁵ is

In embodiments, R⁶ is hydrogen. In embodiments, R⁶ is apolymerase-compatible cleavable moiety. In embodiments, R⁶ is apolymerase-compatible cleavable moiety including an azido moiety. Inembodiments, R⁶ is a polymerase-compatible cleavable moiety including adithiol linker. In embodiments, R⁶ is a polymerase-compatible cleavablemoiety; and the polymerase-compatible cleavable moiety is —CH₂H₃. Inembodiments, the polymerase-compatible cleavable moiety is —NH₂, —CN,—CH₃, C₂-C₆ allyl (e.g., —CH₂—CH═CH₂), methoxyalkyl (e.g., —CH₂—O—CH₃),or —CH₂N₃. In embodiments, R⁶ is —NH₂. In embodiments, R⁶ is —CH₂N₃. Inembodiments, R⁶ is

In embodiments, R⁶ is

In embodiments, R⁶ is

In embodiments, R⁶ is —CH₂—O—CH₃. In embodiments, R⁶ is —NH₂, —CH₂N₃,

or —CH₂—O—CH₃. In embodiments, L³ includes a dithiol linker and R⁶ is—NH₂, —CH₂N₃;

or —CH₂—O—CH₃. In embodiments, L³ is

and R⁶ is —NH₂, —CH₂N₃,

or —CH₂—O—CH₃.

In embodiments, R⁶ is a polymerase-compatible cleavable moiety; and thepolymerase-compatible cleavable moiety is

R^(8C) is hydrogen, CH₃, —CX^(8C) ₃, —CHX^(4C) ₂, —CH₂X^(8C), —OCX^(8C)₃, —OCH₂X^(8C), —OCHX^(8C) ₂, —CN, —OH, —SH, —NH₂, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. The symbol X^(8C) is independently halogen. Inembodiments, R^(8C) is independently unsubstituted phenyl.

In embodiments R⁶ is a polymerase-compatible cleavable moiety; and thepolymerase-compatible cleavable moiety is:

R^(8A) is independently hydrogen, CH₃, —CX³ ₃, —CHX³ ₂, —CH₂X³, —OCX³ ₃,—OCH₂X³, —OCHX³ ₂, —CN, —OH, —SH, —NH₂, substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted alkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroalkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted cycloalkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheterocycloalkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted aryl, or substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted heteroaryl. In embodiments, R_(8A) is independentlyhydrogen, CH₃, —CX³ ₃, —CHX³ ₂, —CH₂X³, —OCX³ ₃, —OCH₂X³, —OCHX³ ₂, —CN,—OH, —SH, —NH₂, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted C₁-C₆ alkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 2 to 6 membered heteroalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted C₃-C₆ cycloalkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 6 memberedheterocycloalkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted phenyl, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 6 membered heteroaryl. R^(8B) isindependently hydrogen, CH₃, —CX⁴ ₃, —CHX⁴ ₂, —OCX⁴ ₃, —OCH₂X⁴, —OCHX⁴₂, —CN, —OH, —SH, —NH₂, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted alkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heteroalkyl, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted cycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heterocycloalkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted aryl, or substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted heteroaryl. Inembodiments, R^(8B) is independently hydrogen, CH₃, —CX⁴ ₃, —CHX⁴ ₂,—CH₂X⁴, —OCX⁴ ₃, —OCH₂X⁴, —OCHX⁴ ₂, —CN, —OH, —SH, —NH₂, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted C₁-C₆ alkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted 2 to 6membered heteroalkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted C₃-C₆ cycloalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 3 to 6 membered heterocycloalkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted phenyl, orsubstituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted 5 to 6membered heteroaryl. R⁹ is independently hydrogen, CH₃, —CX⁵ ₃, —CHX⁵ ₂,—CH₂X⁵, —OCX⁵ ₃, —OCH₂X⁵, —OCHX⁵ ₂, —CN, —OH, —SH, —NH₂, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted alkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted heteroalkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedcycloalkyl, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted heterocycloalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted aryl, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heteroaryl. In embodiments, R⁹ is independentlyhydrogen, —CX⁵ ₃, —CHX⁵ ₂, —CH₂X⁵, —OCX⁵ ₃, —OCH₂X⁵, —OCHX⁵ ₂, —CN, —OH,—SH, —NH₂, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted C₁-C₆ alkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 2 to 6 membered heteroalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted C₃-C₆ cycloalkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 6 memberedheterocycloalkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted phenyl, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 6 membered heteroaryl. le is independentlyhydrogen, CH₃, —CX⁶ ₃, —CHX⁶ ₂, —CH₂X⁶, —OCX⁶ ₃, —OCH₂X⁶, —OCHX⁶ ₂, —CN,—OH, —SH, —NH₂, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted alkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted heteroalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted cycloalkyl, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted heterocycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted aryl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroaryl. In embodiments,R¹⁰ is independently hydrogen, CH₃, —CX⁶ ₃, —CHX⁶ ₂, —CH₂X⁶, —OCX⁶ ₃,—OCH₂X⁶, —OCHX⁶ ₂, —CN, —OH, —SH, —NH₂, substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted C₁-C₆ alkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted 2 to 6 membered heteroalkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted C₃-C₆cycloalkyl, substituted (e.g., substituted with a substitucnt group,size-limited substituent group, or lower substituent group) orunsubstituted 3 to 6 membered heterocycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted phenyl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted 5 to 6 membered heteroaryl.R¹¹ is independently hydrogen, CH₃, —CX⁷ ₃, —CHX⁷ ₂, —CH₂X⁷, —OCX⁷ ₃,—OCH₂X⁷, —OCHX⁷ ₂, —CN, —OH, —SH, —NH₂, substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted alkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroalkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted cycloalkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheterocycloalkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted aryl, or substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted heteroaryl. In embodiments, R¹¹ is independently hydrogen,CH₃, —CX⁷ ₃, —CHX⁷ ₂, —CH₂X⁷, —OCX⁷ ₃, —OCH₂X⁷, —OCHX⁷ ₂, —CN, —OH, —SH,—NH₂, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted C₁-C₆ alkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 2 to 6 membered heteroalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted C₃-C₆ cycloalkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 6 memberedheterocycloalkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted phenyl, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 6 membered heteroaryl. The symbols X³, X⁴,X⁵, X⁶ and X⁷ are independently halogen.

In embodiments, R⁶ is a polymerase-compatible cleavable moiety; and thepolymerase-compatible cleavable moiety is:

wherein, R^(8A) is independently hydrogen, CH₃, —CX³ ₃, —CHX³ ₂, —CH₂X³,—OCX³ ₃, —OCH₂X³, —OCHX³ ₂, —CN, —OH, —SH, —NH₂, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted C₁-C₆ alkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 2 to 6 memberedheteroalkyl, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted C₃-C₆ cycloalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 3 to 6 membered heterocycloalkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted phenyl, orsubstituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted 5 to 6membered heteroaryl; R^(8B) is independently hydrogen, CH₃, —CX⁴ ₃,—CHX⁴ ₂, —OCX⁴ ₃, —OCH₂X⁴, —OCHX⁴ ₂, —CN, —OH, —SH, —NH₂, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted C₁-C₆ alkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted 2 to 6membered heteroalkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted C₃-C₆ cycloalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 3 to 6 membered heterocycloalkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted phenyl, orsubstituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted 5 to 6membered heteroaryl; R⁹ is independently hydrogen, CH₃, —CX⁵ ₃, —CHX⁵ ₂,—CH₂X⁵, —OCX⁵ ₃, —OCH₂X⁵, —OCHX⁵ ₂, —CN, —OH, —SH, —NH₂, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted C₁-C₆ alkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted 2 to 6membered heteroalkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted C₃-C₆ cycloalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 3 to 6 membered heterocycloalkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted phenyl, orsubstituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted 5 to 6membered heteroaryl; R¹⁰ is independently hydrogen, —CX⁶ ₃, —CHX⁶ ₂,—CH₂X⁶, —OCX⁶ ₃, —OCH₂X⁶, —OCHX⁶ ₂, —CN, —OH, —SH, —NH₂, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted C₆ alkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted 2 to 6membered heteroalkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted C₃-C₆ cycloalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 3 to 6 membered heterocycloalkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted phenyl, orsubstituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted 5 to 6membered heteroaryl; R^(H-) is independently hydrogen, CH₃, —CX⁷ ₃,—CHX⁷ ₂, —CH₂X⁷, —OCX⁷ ₃, —OCH₂X⁷, —OCHX⁷ ₂, —CN, —OH, —SH, —NH₂,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted C₁-C₆alkyl, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 2 to 6 membered heteroalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted C₃-C₆ cycloalkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 6 memberedheterocycloalkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted phenyl, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 6 membered heteroaryl; and X³, X⁴, X⁵, X⁶and X⁷ are independently halogen.

In embodiments, R⁶ is a polymerase-compatible cleavable moiety; and thepolymerase-compatible cleavable moiety is:

wherein R^(8A) and R^(8B) are independently hydrogen or unsubstitutedalkyl; R⁹, R¹⁰, and R¹¹ are independently unsubstituted alkyl orunsubstituted heteroalkyl. In embodiments, R⁶ is a polymerase-compatiblecleavable moiety; and the polymerase-compatible cleavable moiety is:

wherein R^(8A) and R^(8B) are independently hydrogen or unsubstitutedC₁-C₄ alkyl; and R⁹, R¹⁰, and R¹¹ are independently unsubstituted C₁-C₆alkyl or unsubstituted 2 to 4 membered heteroalkyl. In embodiments, R⁶is a polymerase-compatible cleavable moiety; and thepolymerase-compatible cleavable moiety is:

wherein R^(8A) and R^(8B) are independently hydrogen; and R⁹, R¹⁰, andR¹¹ are independently unsubstituted C₁-C₆ alkyl or unsubstituted 2 to 4membered heteroalkyl. In embodiments, R⁶ is a polymerase-compatiblecleavable moiety; and the polymerase-compatible cleavable moiety is:

R^(8A) and R^(8B) are independently hydrogen; and R⁹, R¹⁰, and R¹¹ areindependently unsubstituted methyl or unsubstituted methoxy. Inembodiments, R⁶ is a polymerase-compatible cleavable moiety; and thepolymerase-compatible cleavable moiety is:

In embodiments, R⁷ is hydrogen. In embodiments, R⁷ is —OR^(7A); andR^(7A) is hydrogen. In embodiments, R⁷ is —OR^(7A); and R^(7A) is apolymerase-compatible cleavable moiety. In embodiments, R⁷ is —OR^(7A);and R^(7A) is a polymerase-compatible cleavable moiety including anazido moiety. In embodiments, R⁷ is —OR^(7A); and R^(7A) is apolymerase-compatible cleavable moiety including a dithiol linker. Inembodiments, R⁷ is —OR^(7A); R^(7A) is a polymerase-compatible cleavablemoiety; and the polymerase-compatible cleavable moiety is —CH₂N₃. Inembodiments, R⁷ is —OR^(7A); and R^(7A) is a polymerase-compatiblecleavable moiety comprising a dithiol linker, an allyl group, or a2-nitrobenzyl group. In embodiments, R⁷ is —NH₂, —CH₂N₃,

or —CH₂—O—CH₃.

In embodiments, R⁷ is —OR^(7A); R^(7A) is a polymerase-compatiblecleavable moiety; and the polymerase-compatible cleavable moiety is:

In embodiments, R^(7A) is

R^(8C) is hydrogen, CH₃, —CX^(8C) ₃, —CHX^(8C) ₂, —CH₂X^(8C), —OCX^(8C)₃, —OCH₂X^(8C), —OCHX^(8C) ₂, —CN, —OH, —SH, —NH₂, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl. The symbol X^(8C) is independently halogen. Inembodiments, R^(8C) is independently unsubstituted phenyl.

In embodiments, R^(8A), is independently hydrogen, CH₃, —CX³ ₃, —CHX³ ₂,—CH₂X³, —OCX³ ₃, —OCH₂X³, —OCHX³ ₂, —CN, —OH, —SH, —NH₂, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted alkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted heteroalkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedcycloalkyl, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted heterocycloalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted aryl, or substituted (e.g., substituted with asubstitucnt group, size-limited substituent group, or lower substituentgroup) or unsubstituted heteroaryl. In embodiments R^(8A) isindependently hydrogen, CH₃, —CX³ ₃, —CHX³ ₂, —CH₂X³, —OCX³ ₃, —OCH₂X³,—OCHX³ ₂, —CN, —OH, —SH, —NH₂, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted C₁-C₆ alkyl, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted 2 to 6 membered heteroalkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or un substituted C₃-C₆cycloalkyl, substituted (e .g ., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 3 to 6 membered heterocycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted phenyl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted 5 to 6 membered heteroaryl. Inembodiments, R^(8A) is independently hydrogen, deuterium, —C(CH₃)₃,—CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, —CX³ ₃, —CHX³ ₂, —CH₂X³. —CN, or-Ph. In embodiments, R¹¹ is independently hydrogen, deuterium, —C(CH₃)₃,—CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, —CX⁴ ₃, —CHX⁴ ₂, —CH₂X⁴, —CN, or-Ph.

R^(8B) is independently hydrogen, CH₃, —CX⁴ ₃, —CHX⁴ ₂, —CH₂X⁴, —OCX⁴ ₃,—OCH₂X⁴, —OCHX⁴ ₂, —CN, —OH, —SH, —NH₂, substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted alkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroalkyl, substituted(c.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted cycloalkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheterocycloalkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted aryl, or substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted heteroaryl. In embodiments, R^(8B) is independentlyhydrogen, CH₃, —CX⁴ ₃, —CHX⁴ ₂, —CH₂X⁴, —OCX⁴ ₃, —OCH₂X⁴, —OCHX⁴ ₂, —CN,—OH, —SH, —NH₂, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted C₁-C₆ alkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 2 to 6 membered heteroalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted C₃-C₆ cycloalkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 6 memberedheterocycloalkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted phenyl, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 6 membered heteroaryl.

In embodiments, R^(8A) is independently hydrogen, —CH₃, —CX³ ₃, —CHX³ ₂,—CH₂X³, —CN, -Ph, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted alkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted heteroalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted cycloalkyl, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted heterocycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted aryl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroaryl. In embodiments,R^(8B) is independently hydrogen, —CH₃, —CX⁴ ₃, —CHX⁴ ₂, —CH₂X⁴, —CN,-Ph, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted alkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted heteroalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted cycloalkyl, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted heterocycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted aryl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroaryl.

In embodiments, R^(8A) and R^(8B) are independently hydrogen orunsubstituted alkyl. In embodiments, R^(8A) and R^(8B) are independentlyhydrogen or unsubstituted C₁-C₄ alkyl. In embodiments, R^(8A) and R^(8B)are independently hydrogen.

In embodiments, R⁹ is independently hydrogen, CH₃, —CX⁵ ₃, —CHX⁵ ₂,—CH₂X⁵, —OCX⁵ ₃, —OCH₂X⁵, —OCHX⁵ ₂, —CN, —OH, —SH, —NH₂, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted alkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted heteroalkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedcycloalkyl, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted heterocycloalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted aryl, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heteroaryl. In embodiments, R⁹ is independentlyhydrogen, CH₃, —CX⁵ ₃, —CHX⁵ ₂, —CH₂X⁵, —OCX⁵ ₃, —OCH₂X⁵, —OCHX⁵ ₂, —CN,—OH, —SH, —NH₂, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted C₁-C₆ alkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 2 to 6 membered heteroalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted C₃-C₆ cycloalkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 6 memberedheterocycloalkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted phenyl, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 6 membered heteroaryl.

In embodiments, _(R)ic) is independently hydrogen, CH₃, —CX⁶ ₃, —CHX⁶ ₂,—CH₂X⁶, —OCX⁶ ₃, —OCH₂X⁶, —OCHX⁶ ₂, —CN, —OH, —SH, —NH₂, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted alkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted heteroalkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedcycloalkyl, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substitucnt group) orunsubstituted heterocycloalkyl, substituted (e.g., substituted with asubstituent group, size-limited substitucnt group, or lower substituentgroup) or unsubstituted aryl, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heteroaryl. In embodiments, R¹⁰ is independentlyhydrogen, CH₃, —CX⁶ ₃, —CHX⁶ ₂, —CH₂X⁶, —OCX⁶ ₃, —OCH₂X⁶, —OCHX⁶ ₂, —CN,—OH, —SH, —NH₂, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted C₁-C₆ alkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 2 to 6 membered heteroalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted C₃-Co cycloalkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted 3 to 6 memberedheterocycloalkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted phenyl, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted 5 to 6 membered heteroaryl.

In embodiments, R⁹ is independently hydrogen, CH₃, —CX⁵ ₃, —CHX⁵ ₂,—CH₂X⁵, —OCH₃, —NHCH₃, —CN, -Ph, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted alkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heteroalkyl, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted cycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heterocycloalkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted aryl, or substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted heteroaryl; le isindependently hydrogen, CH₃, —CX⁶ ₃, —OCH₃, —SCH₃, —NHCH₃, —CN, -Ph,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted alkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheteroalkyl, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted cycloalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heterocycloalkyl, substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted aryl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroaryl.

In embodiments R¹¹ is independently hydrogen, CH₃, —CX⁷ ₃, —CHX¹ ₂,—CH₂X⁷, —OCX⁷ ₃, —OCH₂X¹, —OCHX⁷ ₂, —CN, —OH, —SH, —NH₂, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted alkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted heteroalkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedcycloalkyl, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted heterocycloalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted aryl, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heteroaryl. In embodiments, _(R)o isindependently hydrogen, —CX⁷ ₃, —CHX⁷ ₂, —CH₂X⁷, —OCX⁷ ₃, —OCH₂X⁷,—OCHX⁷ ₂, —CN, —OH, —SH, —NH₂, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted C₁-C₆ alkyl, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted 2 to 6 membered heteroalkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted C₃-C₆cycloalkyl, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 3 to 6 membered heterocycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substitucnt group, orlower substituent group) or unsubstituted phenyl, or substituted (e.g.,substituted with a substituent group, size-limited substitucnt group, orlower substitucnt group) or unsubstituted 5 to 6 membered heteroaryl.The symbols X³, X⁴, X⁵, X⁶ and X⁷ are independently halogen.

In embodiments, R^(il) is independently hydrogen, CH₃, —CX¹ ₃, —CHX⁷ ₂,—CH₂X⁷, —OCH₃, —SCH₃, —NHCH₃, —CN, -Ph substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted alkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroalkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted cycloalkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheterocycloalkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted aryl, or substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted heteroaryl.

In embodiments, R⁹, R¹⁰, and R¹¹ are independently unsubstituted alkylor unsubstituted heteroalkyl. In embodiments, R⁹, R¹⁰, and R¹¹ areindependently unsubstituted C₁-C₆ alkyl or unsubstituted 2 to 4 memberedheteroalkyl. In embodiments, R⁹, R¹⁰, and R¹¹ are independentlyunsubstituted C₁-C₆ alkyl or unsubstituted 2 to 4 membered heteroalkyl.In embodiments, R⁹, R¹⁰, and R¹¹ are independently unsubstituted methylor unsubstituted methoxy. In embodiments, R^(8A), feu, R⁹, R¹⁰, and R¹¹are independently hydrogen or unsubstituted methyl. In embodiments,R^(8A) and R^(8B) are hydrogen and R⁹, R¹⁰, and R¹¹ are unsubstitutedmethyl.

In embodiments, R⁷ is —OR^(7A); R^(7A) is a polymerase-compatiblecleavable moiety; and the polymerase-compatible cleavable moiety is:

wherein R^(8A) is hydrogen, —CX³ ₃, —CHX³ ₂, —CH₂X³, —OCX³ ₃, —OCH₂X³,—OCHX³ ₂, —CN, —OH, —SH, —NH₂, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted C₁-C₆ alkyl, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted 2 to 6 membered heteroalkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted C₃-C₆cycloalkyl, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 3 to 6 membered heterocycloalkyl, substituted (e.g.,substituted with a substitucnt group, size-limited substitucnt group, orlower substitucnt group) or unsubstituted phenyl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted 5 to 6 membered heteroaryl;R^(8B) is independently hydrogen, CH₃, —CX⁴ ₃, —CHX⁴ ₂, —CH₂X⁴, —OCX⁴ ₃,—OCH₂X⁴, —OCHX⁴ ₂, —CN, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted C₁-C₆ alkyl, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted 2 to 6 membered heteroalkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted C₃-C₆cycloalkyl, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 3 to 6 membered heterocycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted phenyl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted 5 to 6 membered heteroaryl; R⁹is independently hydrogen, CH₃, —CX⁵3_(;)—CHX⁵ ₂, —CH₂X⁵, —OCX⁵ ₃,—OCH₂X⁵, —OCHX⁵ ₂, —CN, —OH, —SH, —NH₂, substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted C₁-C₆ alkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted 2 to 6 membered heteroalkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted C₃-C₆cycloalkyl, substituted (e.g., substituted with a substitucnt group,size-limited substituent group, or lower substituent group) orunsubstituted 3 to 6 membered heterocycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted phenyl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted 5 to 6 membered heteroaryl;R′' is independently hydrogen, CH₃, —CX⁶ ₃; —CHX⁶ ₂, —CH₂X⁶, —OCX⁶ ₃,—OCH₂X⁶; —OCHX⁶ ₂, —CN, —OH, —SH, —NH₂, substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted C₁-C₆ alkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted 2 to 6 membered heteroalkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted C₃-C₆cycloalkyl, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 3 to 6 membered heterocycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted phenyl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted 5 to 6 membered heteroaryl;R¹¹ is independently hydrogen, CH₃, —CX⁷ ₃, —CHX⁷ ₂, —CH₂X⁷, —OCX⁷ ₃,—OCH₂X⁷, —OCHX⁷ ₂, —CN, —OH, —SH, —NH₂, substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted C₁-C₆ alkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted 2 to 6 membered heteroalkyl,substituted (e.g., substituted with a substitucnt group, size-limitedsubstitucnt group, or lower substitucnt group) or unsubstituted C₃-C₆cycloalkyl, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted 3 to 6 membered heterocycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted phenyl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted 5 to 6 membered heteroaryl;and X³, X⁴, X⁵, X⁶ and X⁷ are independently halogen.

In embodiments, R⁷ is —OR^(7A); R^(7A) is a polymerase-compatiblecleavable moiety; and the polymerase-compatible cleavable moiety is:

wherein R^(8A), R^(8B), R⁹, R¹⁰, and R¹¹ are independently hydrogen orunsubstituted methyl. In embodiments, R⁷ is —OR^(7A); R^(7A) is apolymerase-compatible cleavable moiety; and the polymerase-compatiblecleavable moiety is:

In embodiments, R^(7A) is hydrogen. In embodiments, R^(7A) is

In embodiments, R^(7A) is

In embodiments, R^(7A) is

In embodiments, R^(8A) is independently hydrogen, deuterium, —C(CH₃)₃,—CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃,—OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —-SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃,—NHC (CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, —CN, or -Ph.In embodiments, R^(8A) is independently hydrogen, —CH₃, —CX³ ₃, —CN,-Ph, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted alkyl, substituted (e.g., substituted with a substituentgroup, size-limited substituent group, or lower substituent group) orunsubstituted heteroalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted cycloalkyl, substituted (e.g., substituted witha substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted heterocycloalkyl, substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted aryl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroaryl.

In embodiments, R^(8A) is independently hydrogen, C(CH₃)₃, —CH(CH₃)₂,—CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃, —OCH(CH₃)₂, -OCH₂CH₂CH₃, —OCH₂CH₃,—OCH₃, —SC(CH₃)₃, -,-SCH(CH₃)₂, —SCH₂CH₂CH₃, -SCH₂CH₃, —SCH₃,—NHC(CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, —CN, or -Ph.

In embodiments, R^(s)' is independently hydrogen, deuterium, C(CH₃)₃,—CH(CH₃)₂, —CH₂CH₂CH₃, -CH₂CH₃, —CH₃, OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃,—OCH₂CH₃, —OCH₃, —SC(CH₃)₃, -SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃,—NHC(CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, —CN, or -Ph.In embodiments, RS^(B) is hydrogen, —CH₃, —CX⁴ ₃, —CHX⁴ ₂, —CN, -Ph,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted alkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheteroalkyl, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted cycloalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heterocycloalkyl, substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted aryl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroaryl.

In embodiments, R^(8B) is independently hydrogen, —C(CH₃)₃, —CH(CH₃)₂,—CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃,—OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃,—NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, —CN, or -Ph.

In embodiments, —CR⁹R¹⁰R¹¹ is unsubstituted methyl, unsubstituted ethyl,unsubstituted propyl, unsubstituted isopropyl, unsubstituted butyl, orunsubstituted tert-butyl.

In embodiments, R⁹ is independently hydrogen, —C(CH₃)₃, —CH(CH₃)₂,—CH₂CH₂CH₃, —CH₂CH₃, —CH₃, —OC(CH₃)₃; —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃,—OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC (CH₃)₃,—NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, or -Ph. In embodiments, R⁹is hydrogen, —CX⁵ ₃, —CHX⁵ ₂, —OCH₃, —SCH₃, —NHCH₃, —CN, -Ph,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted alkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheteroalkyl, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted cycloalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heterocycloalkyl, substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstitucnt group) or unsubstituted aryl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroaryl.

In embodiments, R¹⁰ is independently hydrogen, —C (CH₃)₃, —CH(CH₃)₂,—CH₂CH₂CH₃—CH₂CH₃, —CH₃, OC(CH₃)₃, —CH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃,—OCH₃, SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃;—NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, or -Ph. le is hydrogen,—CX⁶ ₃, —CHX⁶ ₂, —CH₂X⁶, —OCH₃; —SCH₃, —NHCH₃, —CN, -Ph, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted alkyl, substituted(e.g., substituted with a substituent group, size-limited substituentgroup, or lower substituent group) or unsubstituted heteroalkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedcycloalkyl, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted heterocycloalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted aryl, or substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heteroaryl;

In embodiments, R¹¹ is independently hydrogen, —C(CH₃)₃, —CH(CH₃)₂,—CH₂CH₂CH₃; —CH₂CH₃, —CH₃, OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃,—OCH₃, SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃,—NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, or -Ph. In embodiments,R¹¹ is hydrogen, —CX⁷ ₃, —CHX⁷ ₂, —CH₂X⁷, —OCH₃, —SCH₃, —NHCH₃, —CN, -Phsubstituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstituted alkyl,substituted (e.g., substituted with a substituent group, size-limitedsubstituent group, or lower substituent group) or unsubstitutedheteroalkyl, substituted (e.g., substituted with a substituent group,size-limited substituent group, or lower substituent group) orunsubstituted cycloalkyl, substituted (e.g., substituted with asubstituent group, size-limited substituent group, or lower substituentgroup) or unsubstituted heterocycloalkyl, substituted (e.g., substitutedwith a substituent group, size-limited substituent group, or lowersubstituent group) or unsubstituted aryl, or substituted (e.g.,substituted with a substituent group, size-limited substituent group, orlower substituent group) or unsubstituted heteroaryl.

In embodiments, R¹³ is a fluorescent dye. In embodiments R¹³ is a AlexaFluor® 350 moiety, Alexa Fluor® 405 moiety, Alexa Fluor(®) 430 moiety,Alexa Fluor® 488 moiety, Alexa Fluor® 532 moiety, Alexa Fluor® 546moiety, Alexa Fluor® 555 moiety, Alexa Fluor® 568 moiety, Alexa Fluor®594 moiety, Alexa Fluor® 610 moiety, Alexa Fluor® 633 moiety, AlexaFluor® 635 moiety, Alexa Fluor® 647 moiety, Alexa Fluor® 660 moiety,Alexa Fluor® 680 moiety, Alexa Fluor® 700 moiety, Alexa Fluor® 750moiety, or Alexa Fluor® 790 moiety. In embodiments the detectable moietyis a Alexa Fluor® 488 moiety, Rhodamine 6G (R6G) moiety, ROX ReferenceDye (ROX) moiety, or Cy5 moiety.

In embodiments R¹³ is a FAM™ moiety, TET™ moiety, JOE™ moiety, VIC®moiety, HEX™ moiety, NED™ moiety, PET® moiety, ROX™ moiety, TAMRA™moiety, TET™ moiety, Texas Red® moiety, Alexa Fluor® 488 moiety,Rhodamine 6G (R6G) moiety, ROX Reference Dye (ROX) moiety, Sulfo-Cy5, orCy5 moiety. In embodiments R¹³ is a Rhodamine 6G (R6G) moiety, ROXReference Dye (ROX) moiety, Sulfo-Cy5, or Cy5 moiety.

In embodiments, X¹ is independently —F. In embodiments, X¹ isindependently —Cl. In embodiments, X¹ is independently —Br. Inembodiments, X¹ is independently —I. In embodiments, X² is independently—F. In embodiments, X² is independently —Cl. In embodiments, X² isindependently —Br. In embodiments, X² is independently —I. Inembodiments, X³ is independently —F. In embodiments, X³ is independently—Cl. In embodiments, X³ is independently —Br. In embodiments, X³ isindependently —I. In embodiments, X⁴ is independently —F. Inembodiments, X⁴ is independently —Cl. In embodiments, X⁴ isindependently —Br. In embodiments, X⁴ is independently —I. Inembodiments, X⁵ is independently —F. In embodiments, X⁵ is independently—Cl. In embodiments, X⁵ is independently —Br. In embodiments, X⁵ isindependently —I. In embodiments, X⁶ is independently —F. Inembodiments, X⁶ is independently In embodiments, X⁶ is independently—Br. In embodiments, X⁶ is independently —I. In embodiments, X⁷ isindependently —F. In embodiments, X⁷ is independently —Cl. Inembodiments, X⁷ is independently —Br. In embodiments, X⁷ isindependently —I.

In embodiments, z is an integer from 0 to 20. In embodiments, z is aninteger from 0 to 10. In embodiments, z is an integer from 0 to 15. Inembodiments, z is an integer from 5 to 10. In embodiments, z is 0. Inembodiments, z is 1. In embodiments, z is 2. In embodiments, z is 3. Inembodiments, z is 4. In embodiments, z is 5. In embodiments, z is 6. Inembodiments, z is 7. In embodiments, z is 8. In embodiments, z is 9. Inembodiments, z is 10. in embodiments, z is 11. In embodiments, z is 12.In embodiments, z is 13. In embodiments, z is 14. In embodiments, z is15. In embodiments, z is 16. In embodiments, z is 17. In embodiments, zis 18. In embodiments, z is 19. In embodiments, z is 20.

In embodiments, m is an integer from 1 to 4. In embodiments, m is 1. Inembodiments, m is 2. In embodiments, m is 3. In embodiments, m is 4.

In embodiments, the compound has the formula:

wherein L², R⁵, R^(7A), R^(8A), R^(8B), R⁹, R¹⁰ , and R¹¹ are asdescribed herein, and m is an integer from 1 to 4.

In embodiments, the compound has the formula:

wherein L², R⁵, R^(7A), R^(8A), R^(8B), R⁹, R¹⁰, and R¹¹ are asdescribed herein are as described herein.

In embodiments, the compound has the formula:

wherein L², R⁵, R^(7A), R^(8A), R^(8B), R⁹, R¹⁰, and R¹¹ are asdescribed herein are as described herein.

In embodiments, the compound has the formula:

wherein L², R⁵, and R^(7A) are as described herein are as describedherein.

In embodiments, the compound has the formula:

wherein L², R⁵, R^(8A), R^(8B), R⁹, R¹⁰, and R¹¹ are as described hereinare as described herein and m is an integer from 1 to 4. In embodiments,R^(8A) is hydrogen, deuterium, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃,—CH₃, OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, SC(CH₃)₃,—SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃, —NHCH(CH₃)₂,—NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, —CN, or -Ph. In embodiments, R^(8A) ishydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃,—OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂,—SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃,—NHCH₂CH₃, —NHCH₃—CN, or -Ph. In embodiments, R^(8B) is hydrogen,deuterium, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃,—OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂,—SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃,—NHCH₂CH₃, —NHCH₃—CN, or -Ph. In embodiments, R^(8B) is hydrogen,—C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃, —OCH(CH₃)₂,—OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃,—SCH₂CH₃, —SCH₃, NHC(CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃,—NHCH₃—CN, or -Ph. In embodiments, —CR⁹R¹⁰R¹¹ is unsubstituted methyl,unsubstituted ethyl, unsubstituted propyl, unsubstituted isopropyl,unsubstituted butyl, or unsubstituted tent-butyl. In embodiments, R⁹ ishydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, —OC(CH₃)₃,—OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂,—SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃,—NHCH₂CH₃, —NHCH₃, or -Ph. In embodiments, R¹⁰ is hydrogen, —C(CH₃)₃,—CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃,—OCH₂CH₃, —OCH₃, —S C(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃,—NHC(CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, or -Ph. Inembodiments, R¹¹ is hydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃,—CH₃, —OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —S C (CH₃)₃,—SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃, —NHCH(CH₃)₂,—NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, or -Ph.

In embodiments, the compound has the formula:

wherein L², R⁵, R^(8A), R^(8B), R⁹, R¹⁰, and R¹¹ are as describedherein. In embodiments, R^(8A) is hydrogen, deuterium, —C(CHs)₃,—CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃,—OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃,—NHC(CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃—CN, or -Ph. Inembodiments, R^(8A) is hydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃,—CH₂CH₃, —CH₃, OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃,—SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃,—NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃—CN, or -Ph. In embodiments,R^(8B) is hydrogen, deuterium, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃,—CH₃, OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC (CH₃)₃, —NHCH(CH₃)₂,—NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃—CN, or -Ph. In embodiments, R^(8B) ishydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃,—OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂,—SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC (CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃,—NHCH₂CH₃, —NHCH₃—CN, or -Ph. In embodiments. —CR⁹R¹⁰R¹¹ isunsubstituted methyl, unsubstituted ethyl, unsubstituted propyl,unsubstituted isopropyl, unsubstituted butyl, or unsubstitutedtert-butyl. In embodiments, R⁹ is hydrogen, —C(CH₃)₃, —CH(CH₃)₂,—CH₂CH₂CH₃, —CH₂CH₃, —CH₃, —OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃,—OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃,—NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, —CN, or -Ph. Inembodiments, R¹⁰ is hydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃,—CH₃, —OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃,—SCH(CH₃)₂, —SCH₂ CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃, —NHCH(CH₃)₂,—NHCH₂CH₂ CH₃, —NHCH₂CH₃. —NHCH₃, CN, or -Ph. In embodiments, R¹¹ ishydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, —OC(CH₃)₃,—OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, OCH₃, C(CH₃)₃—SCH(CH₃)₂—SCH₂CH₂CH₃,—SCH₂CH₃ —SCH₃, —NHC(CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃,—NHCH₃, —CN, or -Ph.

In embodiments, the compound has the formula:

wherein L², R⁵, R^(7A), R^(8A), R^(8B), R⁹, R¹⁰, and R¹¹ are asdescribed herein are as described herein.

wherein L² and R⁵ is as described herein.

In embodiments, the compound has formula:

wherein B, R^(7A), R^(8A), R^(8B), R⁹, R¹⁰, and R¹¹ are as describedherein and in is an integer from 1 to 4. In embodiments, R^(8A) ishydrogen, deuterium, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃,OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃,—SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC (CH₃)₃, —NHCH(CH₃)₂,—NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃—CN, or -Ph. In embodiments, R^(8A) ishydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃,—OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂,—SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC (CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃,—NHCH₂CH₃, —NHCH₃—CN, or -Ph. In embodiments, R^(8B) is hydrogen,deuterium, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃,—OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂,—SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC (CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃,—NHCH₂CH₃, —NHCH₃—CN, or -Ph. In embodiments, R^(8B) is hydrogen,—C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃, —OCH(CH₃)₂,—OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃,—SCH₂CH₃, —SCH₃, —NHC (CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃,—NHCH₃—CN, or -Ph. In embodiments, —CR⁹R¹⁰R¹¹ is unsubstituted methyl,unsubstituted ethyl, unsubstituted propyl, unsubstituted isopropyl,unsubstituted butyl, or unsubstituted tert-butyl. In embodiments, R⁹ ishydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂ CH₃, —CH₃, —OC (CH₃)₃,—OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂,—SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃,—NHCH₂CH₃, —NHCH₃, —CN, or -Ph. In embodiments, R¹⁰ is hydrogen,—C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃, —OCH(CH₃)₂,—OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃,—SCH₂CH₃, —SCH₃, —NHC(CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃,—NHCH₃, CN, or -Ph.

In embodiments, R¹¹ is hydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃,—CH₂CH₃, —CH₃, OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃,—SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃,—NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, CN, or -Ph. Inembodiments, R^(7A) is hydrogen. In embodiments R^(7A) is

In embodiments, —R^(7A) is

In embodiments, R^(7A) is

In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, the compound has the formula:

wherein B, R^(7A), R^(8A), R^(8B), R⁹, R¹⁰, and R¹¹ are as describedherein In embodiments, R^(8A) is hydrogen, deuterium, —C(CH₃)₃,—CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃,—OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃,—NHC (CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃—CN, or -Ph. Inembodiments, R^(8A) is hydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃,—CH₂CH₃, —CH₃, OC(CH₃)₃, —OCH(CH)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃,—SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC (CH₃)₃,—NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃—CN, or -Ph. In embodiments,R^(8B) is hydrogen, deuterium, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃,—CH₃, OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃,—SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC (CH₃)₃, —NHCH(CH₃)₂,—NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃—CN, or -Ph. In embodiments, R^(8B) ishydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃,—OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂,—SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC (CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃,—NHCH₂CH₃, —NHCH₃—CN, or -Ph. In embodiments, —CR⁹R¹⁰R¹¹ isunsubstituted methyl, unsubstituted ethyl, unsubstituted propyl,unsubstituted isopropyl, unsubstituted butyl, or unsubstitutedtert-butyl. In embodiments, R⁹ is hydrogen, —C(CH₃)₃, —CH(CH₃)₂,—CH₂CH₂CH₃, —CH₂CH₃, —CH₃, —OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃,—OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC (CH₃)₃,—NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, —CN, or -Ph. Inembodiments, R¹⁰ is hydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃,—CH₃, OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃,—SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃, —NHCH(CH₃)₂,—NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, —CN, or -Ph. In embodiments, R¹¹ ishydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃,—OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂,—SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃,—NHCH₂CH₃, —NHCH₃, —CN, or -Ph. In embodiments, R^(7A) is hydrogen. Inembodiments R^(7A) is

In embodiments, —R^(7A) is

In embodiments, R^(7A) is

In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, the compound has the formula:

wherein B, R⁹, R¹⁰, and R¹¹ are as described herein is are as describedherein.

In embodiments, the compound has the formula:

wherein B and R^(7A) are as described herein are as described herein.

In embodiments, the compound has the formula:

wherein B, R^(8A), R^(8B), R⁹, R¹⁰, and R¹¹ are as described herein andm is an integer from 1 to 4. In embodiments, R^(8A) is hydrogen,deuterium. —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃,—OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, SC(CH₃)₃, —SCH(CH₃)₂,—SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, NHC(CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃,—NHCH₂CH₃, —NHCH₃—CN, or -Ph. In embodiments, R^(8A) is hydrogen,—C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃, —OCH(CH₃)₂,—OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃,—SCH₂CH₃, —SCH₃, —NHC(CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃,—NHCH₃—CN, or -Ph. In embodiments, R^(8B) is hydrogen, deuterium,—C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃, —OCH(CH₃)₂,—OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃,—SCH₂CH₃, —SCH₃, —NHC(CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃,—NHCH₃—CN, or -Ph. In embodiments, R^(8B) is hydrogen, —C(CH₃)₃,—CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃,—OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃,—NHC(CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃—CN, or -Ph. Inembodiments, —CR⁹R¹⁰R¹¹ is unsubstituted methyl, unsubstituted ethyl,unsubstituted propyl, unsubstituted isopropyl, unsubstituted butyl, orunsubstituted ten-butyl. In embodiments, R⁹ is hydrogen, C(CH₃)₃,—CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, —OC(CH₃)₃, —OCH(CH₃)₂,—OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃,—SCH₂CH₃, —SCH₃, —NHC(CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃,—NHCH₃, —CN, or -Ph. In embodiments, 11_¹° is hydrogen, —C(CH₃)₃,—CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃,—OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃,—NHC(CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, or -Ph. Inembodiments, R¹¹ is hydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃,—CH₃, OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃,—SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃, —NHCH(CH₃)₂,—NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, —CN, or -Ph.

In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, the compound has the formula:

wherein B, R^(8A), R^(8B), R⁹, R¹⁰, and R¹¹ are as described herein. Inembodiments, R^(8A) is hydrogen, deuterium, —C(CH₃)₃, —CH(CH₃)₂,—CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃,—OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC (CH₃)₃,—NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, or -Ph. In embodiments,R^(8A) is hydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃,OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃,—SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC (CH₃)₃, —NHCH(CH₃)₂,—NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃—CN, or -Ph. In embodiments, R^(8B) ishydrogen, deuterium, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃,OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃,—SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC (CH₃)₃, —NHCH(CH₃)₂,—NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃—CN, or -Ph. In embodiments, R^(8B) ishydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃,—OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂,—SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC (CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃,—NHCH₂CH₃, —NHCH₃—CN, or -Ph. In embodiments, —CR⁹R¹⁰R¹¹ isunsubstituted methyl, unsubstituted ethyl, unsubstituted propyl,unsubstituted isopropyl, unsubstituted butyl, or unsubstitutedtert-butyl. In embodiments, R⁹ is hydrogen, —C(CH₃)₃, —CH(CH₃)₂,—CH₂CH₂CH₃, —CH₂CH₃, —CH₃, —OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃,—OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC (CH₃)₃,—NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃. —CN, or -Ph. Inembodiments, R¹⁰ is hydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃,—CH₃, OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃,—SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃, —NHCH(CH₃)₂,—NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, —CN, or -Ph. In embodiments, R¹¹ ishydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃,—OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂,—SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃,—NHCH₂CH₃, —NHCH₃, —CN, or -Ph. In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, the compound has the formula:

wherein B, R⁹, R¹⁰, and R¹¹ are as described herein are as describedherein. In embodiments, B is

In embodiments, the compound has the formula:

wherein B is as described herein. In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, the compound has the formula:

wherein R^(7A), R^(8A), R^(8B), R⁹, R¹⁰, and R¹¹ are as described hereinand m is an integer from 1 to 4. In embodiments, R^(8A) is hydrogen,deuterium, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃,—OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂,—SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃,—NHCH₂CH₃, —NHCH₃—CN, or -Ph. In embodiments, R^(8A) is hydrogen,—C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃, —OCH(CH₃)₂,—OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃,—SCH₂CH₃, —SCH₃, —NHC (CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃,—NHCH₃—CN, or -Ph. In embodiments, R^(8B) is hydrogen, deuterium,—C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃, —OCH(CH₃)₂,—OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃,—SCH₂CH₃, —SCH₃, —NHC (CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃,—NHCH₃—CN, or -Ph. In embodiments, R^(8B) is hydrogen, —C(CH₃)₃,—CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃, —OCH(CH₃)₂. —OCH₂CH₂CH₃,—OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃,—NHC (CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃—CN, or -Ph. Inembodiments, —CR⁹R¹⁰R¹¹ is unsubstituted methyl, unsubstituted ethyl,unsubstituted propyl, unsubstituted isopropyl, unsubstituted butyl, orunsubstituted tert-butyl. In embodiments, R⁹ is hydrogen, —C(CH₃)₃,—CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, —OC(CH₃)₃, —OCH(CH₃)₂,—OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃,—SCH₂CH3—SCH₃, —NHC(CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃,—CN, or -Ph. In embodiments, R¹⁰ is hydrogen, —C(CH₃)₃, CH(CH₃)₂,—CH₂CH₂CH₃,—CH₂CH₃, —CH₃, OC(CH₃)₃, CH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃—CH₃,—SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃,—NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, or -Ph. In embodiments,R^(H) is hydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃,OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃,—SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃, —NHCH(CH₃)₂,—NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, or -Ph. In embodiments, B is

In embodiments. B is

In embodiments, B is

In embodiments, B is

In embodiments. B is

In embodiments, the compound has the formula:

wherein R^(7A), R^(8A), R^(8B), R⁹, R¹⁰, and R¹¹ are as describedherein. In embodiments, R^(8A) is hydrogen, denterium, —C(CH₃)₃,—CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃,—OCH₂CH₃, —OCH₃, SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃,NHC(CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃—CN, or -Ph. Inembodiments, R^(8A) is hydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃,—CH₂CH₃, —CH₃, OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃,SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, NHC(CH₃)₃,—NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃—CN, or -Ph. In embodiments,R^(8B) is hydrogen, deuterium, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃,—CH₃, OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, SC(CH₃)₃,—SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, NHC(CH₃)₃, —NHCH(CH₃)₂,—NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃—CN, or -Ph. In embodiments, R^(8B) ishydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃,—OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂,—SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃,—NHCH₂CH₃, —NHCH₃—CN, or -Ph. In embodiments, —CR⁹R¹° R¹¹ isunsubstituted methyl, unsubstituted ethyl, unsubstituted propyl,unsubstituted isopropyl, unsubstituted butyl, or unsubstitutedtert-butyl. In embodiments, R⁹ is hydrogen, C(CH₃)₃, —CH(CH₃)₂,—CH₂CH₂CH₃, —CH₂CH₃, —CH₃, —OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃,—OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC (CH₃)₃,—NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, or -Ph. In embodiments,R¹⁰ is hydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃,OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃,—SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃, —NHCH(CH₃)₂,—NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, —CN, or -Ph. In embodiments, R^(H) ishydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃,—OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂,—SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃,—NHCH₂CH₃, —NHCH₃, —CN, or -Ph. In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, the compound has the formula:

In embodiments, the compound has the formula:

wherein R^(7A), R⁹, R¹⁰, and R¹¹ are as described herein.

In embodiments, the compound has the formula:

wherein R^(7A) is as described herein.

In embodiments, the compound has the formula:

wherein R^(8A), R^(8B), R⁹, R¹⁰ , and R¹¹ are as described herein and mis an integer from 1 to 4. In embodiments, R^(8A) is hydrogen,denterium, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃,—OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂,—SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC (CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃,—NHCH₂CH₃, —NHCH₃—CN, or -Ph. In embodiments, R^(8A) is hydrogen,—C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃, —OCH(CH₃)₂,—OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃,—SCH₂CH₃, —SCH₃, —NHC (CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃,—NHCH-CN₃, or -Ph. In embodiments, R^(8B) is hydrogen, denterium,—C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃, —OCH(CH₃)₂,—OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃,—SCH₂CH₃, —SCH₃, —NHC (CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃,—NHCH₃—CN, or -Ph. In embodiments, R^(8B) is hydrogen, —C(CH₃)₃,—CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃,—OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃,—NHC (CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃—CN, or -Ph. Inembodiments, —CR⁹R¹⁰R¹¹ is unsubstituted methyl, unsubstituted ethyl,unsubstituted propyl, unsubstituted isopropyl, unsubstituted butyl, orunsubstituted tert-butyl. In embodiments, R⁹ is hydrogen, —C(CH₃)₃,—CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, —OC(CH₃)₃, —OCH(CH₃)₂,—OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃,—SCH₂CH₃, —SCH₃, —NHC (CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃,—NHCH₃, or -Ph. In embodiments, R¹⁰ is hydrogen, —C(CH₃)₃, —CH(CH₃)₂,—CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃,—OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃,—NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, —CN, or -Ph. Inembodiments, R¹¹ is hydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃,—CH₃, OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃,—SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₂, —SCH₃, —NHC(CH₃)₃, —NHCH(CH₃)₂,—NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, —CN, or -Ph. In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, the compound has the formula:

wherein R^(8A), R^(8B), R⁹, R¹⁰ and R¹¹ are as described herein. Inembodiments, R^(8A) is hydrogen, deuterium, —C(CH₃)₃, —CH(CH₃)₂,—CH₂CH₂CH₃. —CH₂CH₃, —CH₃, OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃,—OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC (CH₃)₃,—NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃—CN, or -Ph. In embodiments,R^(8A) is hydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃,OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃,—SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC (CH₃)₃, —NHCH(CH₃)₂,—NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃—CN, or -Ph. In embodiments, R^(8B) ishydrogen, denterium, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃,OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃,—SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC (CH₃)₃, —NHCH(CH₃)₂,—NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃—CN, or -Ph. In embodiments, R^(8B) ishydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃,—OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —SCH(CH—SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC (CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃,—NHCH₂CH₃, —NHCH₃—CN, or -Ph. In embodiments, —CR⁹R¹⁰R¹¹ isunsubstituted methyl, unsubstituted ethyl, unsubstituted propyl,unsubstituted isopropyl, unsubstituted butyl, or unsubstitutedtert-butyl. In embodiments, R⁹ is hydrogen, —C(CH₃)₃, —CH(CH₃)₂,—CH₂CH₂CH₃, —CH₂CH₃, —CH₃, —OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃,—OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC (CH₃)₃,—NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, —CN, or -Ph. Inembodiments, R⁹ is hydrogen, —C(CH₃)₃, CH(CH₃)₂, —CH₂CH₂CH₃—CH₂CH₃,—CH₃, —OC(CH₃)₃ CH(CH₃)₂ —OCH₂CH₂CH₃, —OCH₂CH₃—CH₃, —SC(CH₃)₃,—SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃, —NHCH(CH₃)₂,—NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, —CN, or -Ph. In embodiments, R¹¹ ishydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃,—OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂,—SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃,—NHCH₂CH₃, —NHCH₃, —CN, or -Ph.

In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, the compound has the formula:

wherein R⁹, R¹⁰, and R¹¹ are as described herein.

In embodiments, the compound has the fommla:

In embodiments, the compound has the formula:

wherein B, R⁵, R^(7A), R^(8A), R^(8B), R⁹, R¹⁰, and R¹¹ are as describedherein and m is art integer from 1 to 4.

In embodiments, the compound has the formula:

wherein B, R⁵, R^(7A), R^(8A), R^(8B), R⁹, R¹⁰, and R¹¹ are as describedherein. In embodiments, R^(8A) is hydrogen, denterium, —C(CH₃)₃,—CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, —OC(CH₃)₃, —OCH(CH₃)₂,—OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃,—SCH₂CH₃, —SCH₃, —NHC (CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃,—NHCH₃—CN, or -Ph. In embodiments, R^(BA) is hydrogen, —C(CH₃)₃,—CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃,—OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃,—NHC (CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃—CN, or -Ph. Inembodiments, R^(8B) is hydrogen, denterium, —C(CH₃)₃, —CH(CH₃)₂,—CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃,—OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃,—NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃—CN, or -Ph. In embodiments,R^(8B) is hydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃,OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃,—SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC (CH₃)₃, —NHCH(CH₃)₂,—NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃—CN, or -Ph. In embodiments, —CR⁹R¹⁰R¹¹is unsubstituted methyl, unsubstituted ethyl, unsubstituted propyl,unsubstituted isopropyl, unsubstituted butyl, or unsubstitutedtert-butyl. In embodiments, R⁹ is hydrogen, —C(CH₃)₃, —CH(CH₃)₂,—CH₂CH₂CH₃, —CH₂CH₃, —CH₃, —OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃,—OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃,—NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃. —CN, or -Ph. Inembodiments, R¹⁰ is hydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃,—CH₃, OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃,—SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃, —NHCH(CH₃)₂,—NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, —CN, or -Ph. In embodiments, R¹¹ ishydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃,—OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂,—SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃,—NHCH₂CH₃, —NHCH₃, —CN, or -Ph. In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, R⁵ is

In embodiments, the compound has the formula:

wherein B, R⁵, R^(7A), R⁹, R¹⁰, and R¹¹ are as described herein. Inembodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments. B is

In embodiments, R⁵ is

In embodiments, the compound has the formula:

wherein B, R^(7A) and R⁵ are as described herein. In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, R⁵ is

In embodiments, the compound has the fbrmula:

wherein B, R⁵, R^(8A), R^(8B), R⁹, R¹⁰, and R¹¹ are as described hereinand in is an integer from 1 to 4. In embodiments, R^(8A) is hydrogen,denterium, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃,—OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂,—SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃,—NHCH₂CH₃, —NHCH₃—CN, or -Ph. In embodiments, R^(8A) is hydrogen,—C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃, —OCH(CH₃)₂,—OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃,—SCH₂CH₃, —SCH₃, —NHC(CH₃)₃, -NIICH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃,—NHCH₃—CN, or -Ph. In embodiments, R^(8B) is hydrogen, deuterium,C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃, —OCH(CH₃)₂,—OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃,—SCH₂CH₃, —SCH₃, —NHC(CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃,—NHCH₃—CN, or -Ph. In embodiments, R^(8B) is hydrogen, —C(CH₃)₃,—CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃,—OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃—SCH₃,—NHC(CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃—CN, or -Ph. Inembodiments, CR⁹R¹⁰R¹¹ is unsubstituted methyl, unsubstituted ethyl,unsubstituted propyl, unsubstituted isopropyl, unsubstituted butyl, orunsubstituted tert-butyl. in embodiments, R⁹ is hydrogen, —C(CH₃)₃,—CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, —OC(CH₃)₃, —OCH(CH₃)₂,—OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃,—SCH₂CH₃, —SCH₃, —NHC(CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃,—NHCH₃, or -Ph. In embodiments, R¹⁰ is hydrogen, —C(CH₃)₃, —CH(CH₃)₂,—CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃,—OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃,—NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, —CN, or -Ph. Inembodiments, R¹¹ is hydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃,—CH₃, OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃,—SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃, —NHCH(CH₃)₂,—NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, —CN. or -Ph. In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, R⁵ is

In embodiment the the o pound has the formula:

wherein B. R⁵, R^(8A), R^(8B), R⁹, R¹⁰, and R¹¹ are as described hereinare as described herein. In embodiments, R^(8A) is hydrogen, deuterium,—C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃, —OCH(CH₃)₂,—OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃,—SCH₂CH₃, —SCH₃, —NHC(CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃,—NHCH₃—CN, or -Ph. In embodiments, R^(8A) is hydrogen, —C(CH₃)₃,—CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃, —OCH(CH₃)₂,—OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃,—SCH₂CH₃, —SCH₃, —NHC (CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃,—NHCH₃—CN, or -Ph. In embodiments, R^(8B) is hydrogen, denterium,—C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃, —OCH(CH₃)₂,—OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃,—SCH₂CH₃, —SCH₃, —NHC (CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃,—NHCH₃—CN, or -Ph. In embodiments, R^(8B) is hydrogen, —C(CH₃)₃,—CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃,—OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃,—NHC (CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃—CN, or -Ph. Inembodiments, —CR⁹R¹⁰R¹¹ is unsubstituted methyl, unsubstituted ethyl,unsubstituted propyl, unsubstituted isopropyl, unsubstituted butyl, orunsubstituted tert-butyl. In embodiments, R⁹ is hydrogen, —C(CH₃)₃,—CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, —OC(CH₃)₃, —OCH(CH₃)₂,—OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃,—SCH₂CH₃, —SCH₃, —NHC (CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃,—NHCH₃. —CN, or -Ph. In embodiments, R¹⁰ is hydrogen, —C(CH₃)₃,—CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃,—OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃,—NHC(CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, —CN, or -Ph.In embodiments, R¹¹ is hydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃,—CH₂CH₃, —CH₃, OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃,—SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃,—NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, —CN, or -Ph. Inembodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, B is

In embodiments, R⁵ is

In embodiments, the compound as the formula:

wherein B, R⁵, R⁹, R¹⁰, and R¹¹ are as described herein, in embodiments,R⁵ is

In embodiments, the compound has the formula:

wherein B and R⁵ are as described herein. In embodiments, R⁵ is

In embodiments, the compound has the formula:

wherein R^(7A) is as described herein.

In embodiments the detectable label is a Alexa Fluor® 350 moiety, AlexaFluor® 405 moiety, Alexa Fluor® 430 moiety, Alexa Fluor® 488 moiety,Alexa Fluor® 532 moiety, Alexa Fluor® 546 moiety, Alexa Fluor® 555moiety, Alexa Fluor® 568 moiety, Alexa Fluor® 594 moiety, Alexa Fluor®610 moiety, Alexa Fluor® 633 moiety, Alexa Fluor® 635 moiety, AlexaFluor® 647 moiety, Alexa Fluor® 660 moiety, Alexa Fluor® 680 moiety,Alexa Fluor® 700 moiety, Alexa Fluor® 750 moiety, or Alexa Fluor® 790moiety. In embodiments the detectable moiety is a Alexa Fluor® 488moiety, Rhodamine 6G (R6G) moiety, ROX Reference Dye (ROX) moiety, orCy5 moiety.

In embodiments the detectable moiety is a FAM™ moiety, TET™ moiety, JOE™moiety, VIC® moiety, HEX™ moiety, NED™ moiety, PET® moiety, ROX™ moiety,TAMRA™ moiety, TET™ moiety, Texas Red® moiety, Alexa Fluor® 488 moiety,Rhodamine 6G (R6G) moiety, ROX Reference Dye (ROX) moiety, Sulfo-Cy5, orCy5 moiety.

In embodiments, the compound has the formula:

In an aspect is provided a compound having the formula:R^(12z)-L^(4z)-R¹³, L^(4z) is a covalent linker. R^(12z) is acomplementary anchor moiety reactive group. R¹³ is a detectable label.In embodiments, the compound has the formula:

wherein R^(12z) is as described herein and z is an integer from 0 to 20.

In embodiments, R^(12z) is

a streptavidin moiety, or

In embodiments, R^(12z) is

In embodiments, R^(12z) is

In embodiments, R^(12z) is

In embodiments, R^(12z) is

In embodiments, R^(12z) is

In embodiments, R^(12z) is

In embodiments, R^(12z) is a streptavidin moiety. In embodiments,R^(12z) is

In embodiments, R¹³ is a fluorescent dye. In embodiments, R¹³ includes afluorescence resonance energy transfer donor fluorescent dye. Inembodiments, R¹³ includes a fluorescence resonance energy transferacceptor fluorescent dye. In embodiments, R¹³ includes a fluorescenceresonance energy transfer donor and acceptor fluorescent dye pairconnected by a linker.

In embodiments, R¹³ includes a fluorescence resonance energy transferdonor and acceptor fluorescent dye pair connected by a linker andseparated by 0.1 nm to 10 nm.

In embodiments, R¹³ is

In embodiments, the compound has the formula:

wherein z1 is an integer from 0 to 50,

In an aspect is provided a compound of the formula: R^(12z)-R¹⁴, R^(12z)is a complementary anchor moiety reactive group. R¹⁴ is R¹⁵-substitutedalkyl, R¹⁵-substituted heteroalkyl, R¹⁵-substituted cycloalkyl,R¹⁵-substituted heterocycloalkyl, R¹⁵-substituted aryl, orR¹⁵-substituted heteroaryl. R¹⁵ is independently R¹⁶-substituted alkyl,R¹⁶-substituted heteroalkyl, R¹⁶-substituted cycloalkyl, R¹⁶-substitutedheterocycloalkyl, R¹⁶-substituted aryl, R¹⁶-substituted heteroaryl, or adetectable dye. R₁₆ is independently R¹⁷-substituted alkyl,R¹⁷-substituted heteroalkyl, R¹⁷-substituted cycloalkyl, substitutedheterocycloalkyl, R¹⁷-substituted aryl, R¹⁷-substituted heteroaryl, or adetectable dye. R¹⁷ is independently R¹⁸-substituted alkyl,R¹⁸-substituted heteroalkyl, R¹⁸-substituted cycloalkyl, R¹⁸-substitutedheterocycloalkyl, R¹⁸-substituted aryl, R¹⁸-substituted heteroaryl, or adetectable dye .R¹⁸ is a detectable dye. R¹⁴ is substituted with aplurality of R¹⁵ moieties, R¹⁵ is substituted with a plurality of R¹⁶moieties, and R¹⁶ is substituted with a plurality of R¹⁷ moieties.

In embodiments, R^(12z) is

a streptavidin moiety, or

In embodiments, R^(12z) is

In embodiments, R^(12z) is

In embodiments, R^(12z) is

In embodiments, R^(12z) is

In embodiments, R^(12z) is

In embodiments, R^(12z) is

In embodiments, R^(l2z) is

In embodiments, R^(12z) is a streptavidin moiety. In embodiments,R^(l2)z is

In embodiments, the detectable dye is a fluorescent dye. In embodiments,the detectable dye includes a fluorescence resonance energy transferdonor fluorescent dye. In embodiments, the detectable dye includes afluorescence resonance energy transfer acceptor fluorescent dye. Inembodiments, the detectable dye includes a fluorescence resonance energytransfer donor and acceptor fluorescent dye pair connected by a linker.In embodiments, the detectable dye includes a fluorescence resonanceenergy transfer donor and acceptor fluorescent dye pair connected by alinker and separated by 0.1 nm to 10 nm.

In embodiments, the detectable dye is

In embodiments, the compound has the formula:

In embodiments, the compound has the formula:

wherein R^(12z) is as described herein.

III. Methods of Use

Provided in an aspect is a method for sequencing a nucleic acid,including: (i) incorporating in series with a nucleic acid polymerase,within a reaction vessel, one of four different labeled nucleotideanalogues into a primer to create an extension strand, wherein theprimer is hybridized to the nucleic acid and wherein each of the fourdifferent labeled nucleotide analogues include a unique detectablelabel; (ii) detecting the unique detectable label of each incorporatednucleotide analogue, so as to thereby identify each incorporatednucleotide analogue in the extension strand, thereby sequencing thenucleic acid. Each of the four different labeled nucleotide analoguesare of the structure as described herein, including embodiments, whereinin the first of the four different labeled nucleotide analogues, B is athymidine or uridine hybridizing base; in the second of the fourdifferent labeled nucleotide analogues, B is an adenosine hybridizingbase; in the third of the four different labeled nucleotide analogues, Bis an guanosine hybridizing base; and in the fourth of the fourdifferent labeled nucleotide analogues, B is an cytosine hybridizingbase.

In embodiments, the method further includes further including, aftereach of the incorporating steps, adding to the reaction vessel fourdifferent unlabeled nucleotide analogues, wherein each of the fourdifferent unlabeled nucleotide analogues are of the structure asdescribed herein, including embodiments, wherein in the first of thefour different unlabeled nucleotide analogues, B is a thymidine oruridine hybridizing base; in the second of the four different unlabelednucleotide analogues, B is an adenosine hybridizing base; in the thirdof the four different unlabeled nucleotide analogues, B is a guanosinehybridizing base; and in the fourth of the four different unlabelednucleotide analogues, B is a cytosine hybridizing base.

In embodiments, at least one of the four different labeled nucleotideanalogues is an orthogonally cleavable labeled nucleotide analogueincluding a cleavable moiety, the orthogonally cleavable labelednucleotide analogue having the structure as described herein, andwherein the method further includes, after each of the incorporatingsteps, adding to the reaction vessel a cleaving reagent capable ofcleaving the cleavable moiety. In embodiments, the cleaving reagent isan acid, base, oxidizing agent, reducing agent, Pd(0),tris-(2-carboxyethyl)phosphine, dilute nitrous acid, fluoride,tris(3-hydroxypropyl)phosphine), sodium dithionite (Na₂S₂O₄), orhydrazine (N₂H₄). In embodiments, the cleaving reagent includes an acid,base, oxidizing agent, reducing agent, Pd(0),tris-(2-carboxyethyl)phosphine, dilute nitrous acid, fluoride,tris(3-hydroxypropyl)phosphine), sodium dithionite (Na₂S₂O₄), orhydrazine (N₂H₄).

In another aspect is a method for sequencing a nucleic acid, including:(i) incorporating in series with a nucleic acid polymerase, within areaction vessel, one of four different nucleotide analogues into aprimer to create an extension strand, wherein the primer is hybridizedto the nucleic acid and wherein three of the four different nucleotideanalogues are different labeled nucleotide analogues each including aunique detectable label and one of the four different nucleotideanalogues is a different unlabeled nucleotide analogue; (ii) detectingthe presence or absence of the unique detectable label of eachincorporated nucleotide analogue, so as to thereby identify eachincorporated nucleotide analogue in the extension strand, therebysequencing the nucleic acid; and wherein each of the four differentlabeled nucleotide analogues are of the structure as described herein,including embodiments, wherein in the first of the four differentlabeled nucleotide analogues, B is a thymidine or uridine hybridizingbase; in the second of the four different labeled nucleotide analogues.B is an adenosine hybridizing base; in the third of the four differentlabeled nucleotide analogues, B is a guanosine hybridizing base; and inthe fourth of the four different labeled nucleotide analogues, B is acytosine hybridizing base.

In embodiments, the method further including, after each of theincorporating steps, adding to the reaction vessel four differentunlabeled nucleotide analogues, wherein each of the four differentunlabeled nucleotide analogues are of the structure as described herein,including embodiments, wherein in thc first of thc four differentunlabeled nucleotide analogues, B is a thymidinc or uridine hybridizingbase; in the second of the four different unlabeled nucleotideanalogues, B is an adenosine hybridizing base; in the third of the fourdifferent unlabeled nucleotide analogues, B is a guanosine hybridizingbase; and in the fourth of the four different unlabeled nucleotideanalogues, B is a cytosine hybridizing base.

In embodiments, at least one of the three different labeled nucleotideanalogues is an orthogonally cleavable labeled nucleotide analogueincluding a cleavable moiety, the orthogonally cleavable labelednucleotide analogue having the structure as described herein, includingembodiments, and wherein the method further includes, after each of theincorporating steps, adding to the reaction vessel a cleaving reagentcapable of cleaving the cleavable moiety. In embodiments, the cleavingreagent is an acid, base, oxidizing agent, reducing agent, Pd(0),tris-(2-carboxyethyl)phosphine, dilute nitrous acid, fluoride,tris(3-hydroxypropyl)phosphine), sodium dithionite (Na₂S₂O₄), orhydrazine (N₂H₄). In embodiments, the cleaving reagent includes an acid,base, oxidizing agent, reducing agent, Pd(0),tris-(2-carboxyethyl)phosphine, dilute nitrous acid, fluoride,tris(3-hydroxypropyl)phosphine), sodium dithionite (Na₂S₂O₄), orhydrazine (N₂H₄).

In an aspect is provided a method of incorporating a nucleotide analogueinto a primer, the method including combining a polymerase, a primerhybridized to nucleic acid template and a nucleotide analogue within areaction vessel and allowing the polymerase to incorporate thenucleotide analogue into the primer thereby forming an extended primer,wherein the nucleotide analogue is of the structure as described herein,including embodiments.

In embodiments, L² is a cleavable moiety and R⁵ is a detectable label,the method further including, after the incorporating, cleaving thecleavable moiety with a cleaving reagent. In embodiments, the cleavingreagent is an acid, base, oxidizing agent, reducing agent, Pd(0),tris-(2-carboxyethyl)phosphine, dilute nitrous acid, fluoride,tris(3-hydroxypropyl)phosphine), sodium dithionite (Na₂S₂O₄), orhydrazine (N₂H₄). In embodiments, the cleaving reagent includes an acid,base, oxidizing agent, reducing agent, Pd(0),tris-(2-carboxyethyl)phosphine, dilute nitrous acid, fluoride,tris(3-hydroxypropyl)phosphine), sodium dithionite (Na₂S₂O₄), orhydrazine (N₂H₄).

In embodiments, R⁵ is anchor moiety, the method further including, afterthe incorporating, labeling the nucleotide analog with a detectablelabel. In embodiments, R⁵ is an affinity anchor moiety. In embodiments,the labeling includes adding to the reaction vessel a compound havingthe formula R¹²-L⁴-R¹³, wherein R¹² is a complementary affinity anchormoiety binder; R¹³ is a detectable label; and L⁴ is a covalent linker.

In embodiments, R⁵ is a chemically reactive anchor moiety. Inembodiments, R⁵ is a bioconjugate reactive group.

In embodiments, the labeling includes adding to the reaction vessel acompound having the formula R^(12z)-L^(4z)-R¹³, wherein R^(12z) is acomplementary anchor moiety reactive group; R¹¹ is a detectable label;and L^(4z) is a covalent linker. In embodiments, R^(12z)-L^(4z)_R¹³ hasthe structure as described herein. In embodiments, L^(4z) is a cleavablelinker.

In embodiments, the method further including, after the incorporating,cleaving the cleavable moiety with a cleaving reagent. In embodiments,the cleaving reagent is an acid, base, oxidizing agent, reducing agent,Pd(0), tris-(2-carboxycthyl)phosphine, dilute nitrous acid, fluoride,tris(3-hydroxypropyl)phosphine), sodium dithionite (Na₂S₂O₄), orhydrazine (N₂H₄). In embodiments, the cleaving reagent includes an acid,base, oxidizing agent, reducing agent, Pd(0),tris-(2-carboxyethyl)phosphine, dilute nitrous acid, fluoride,tris(3-hydroxypropyl)phosphine), sodium dithionite (Na₂S₂O₄), orhydrazine (N₂H₄).

In embodiments, the method further including, after the incorporating,adding to the reaction vessel an unlabeled nucleotide analogue includinga 3′-polymerase-compatible cleavable moiety.

In embodiments, the method forms part of a sequencing by synthesismethod.

In embodiments, the ratio of fluorescently labeled to unlabeledcompounds described herein (e.g., nucleotide reversible terminators) isabout 1:9 to about 9:1. (See FIGS. 27A-27B)

In an embodiment, a method of sequencing nucleic acids comprisingaddition of the DNA polymerase and a labeled nucleotide analogue to theprimed DNA template to enable the incorporation of the complementarylabeled nucleotide analogue into the growing DNA strand and identifyingthe labeled nucleotide directly or through indirect labeling, so as tosequence the nucleic acid.

In an embodiment, a method of sequencing nucleic acid comprising: a)providing a nucleic acid template hybridized to a primer; b) extendingthe primer hybridized to said nucleic acid template with a labelednucleotide or nucleotide analogue, wherein said labeled nucleotide ornucleotide analogue has the label linked to the base and apolymerase-compatible cleavable blocking group on the 3′-hydroxyl group;and c) identifying the labeled nucleotide, so as to sequence the nucleicacid.

In an embodiment, a method of simultaneously sequencing a plurality ofdifferent nucleic acids, comprising: a) growing a plurality ofdouble-stranded DNA, each of which comprises one of said DNA strands, byincorporating a labeled nucleotide; and b) identifying each labelednucleotide, so as to simultaneously sequence the plurality of differentnucleic acids.

In another embodiment said labeled nucleotide has the label linked tothe base and a polymerase-compatible cleavable blocking group on the3′-hydroxyl group.

For any of the above three embodiments, wherein:

1. The polymerase-compatible cleavable blocking group comprises adithiol linker.

2. The polymerase-compatible cleavable blocking group comprises an azidomoiety.

3. The polymerase-compatible cleavable blocking group comprises—CH₂SS—R, —CH₂N₃, allyl, 2-nitrobenzyl, cyanoethyl, or azo.

4. The polymerase-compatible cleavable blocking group is a dithiolhaving the following structure:

-   -   R^(8A) is hydrogen, —CH₃, —CX³ ₃, —CHX³ ₂, —CH₂X³, —CN, -Ph,        substituted or unsubstituted alkyl, substituted or unsubstituted        heteroalkyl, substituted or unsubstituted cycloalkyl,        substituted or unsubstituted heterocycloalkyl, substituted or        unsubstituted aryl, or substituted or unsubstituted heteroaryl;    -   R^(8B) is hydrogen, —CH₃, —CX⁴ ₃, —CHX⁴ ₂, —CH₂X⁴, —CN, -Ph,        substituted or unsubstituted alkyl, substituted or unsubstituted        heteroalkyl, substituted or unsubstituted cycloalkyl,        substituted or unsubstituted heterocycloalkyl, substituted or        unsubstituted aryl, or substituted or unsubstituted heteroaryl;    -   R⁹ is hydrogen, —CX¹ ₃, —CHX⁵ ₂, —CH₂X⁵, —OCH₃, —SCH₃, —NHCH₃,        —CN, -Ph, substituted or unsubstituted alkyl, substituted or        unsubstituted heteroalkyl, substituted or unsubstituted        cycloalkyl, substituted or unsubstituted heterocycloalkyl,        substituted or unsubstituted aryl, or substituted or        unsubstituted heteroaryl;    -   R¹⁰ is hydrogen, —CX⁶ ₃, —CHX⁶ ₂, —CH₂X⁶, —OCH₃, —SCH₃, —NHCH₃,        —CN, -Ph, substituted or unsubstituted alkyl, substituted or        unsubstituted heteroalkyl, substituted or unsubstituted        cycloalkyl, substituted or unsubstituted heterocycloalkyl,        substituted or unsubstituted aryl, or substituted or        unsubstituted heteroaryl;    -   R¹¹ is hydrogen, —CX⁷ ₃, —CHX⁷ ₂, —CH₂X⁷, —OCH₃, —SCH₃, —NHCH₃,        —CN, -Ph substituted or unsubstituted alkyl, substituted or        unsubstituted heteroalkyl, substituted or unsubstituted        cycloalkyl, substituted or unsubstituted heterocycloalkyl,        substituted or unsubstituted aryl, or substituted or        unsubstituted heteroaryl; and    -   X³, X⁴, X⁵, X⁶ and X⁷ are independently halogen.

5. The label is attached to the base via a cleavable linker.

6. The labeled nucleotide has the label attached to the 5 or 7 positionof the base via a cleavable linker.

7. The nucleotide analogue comprises a deazapurine base.

8. The said cleavable linker is indicated by L3 in the followingstructure:

wherein

-   -   B is a base;    -   L³ is a cleavable linker;    -   R³ is —OH, monophosphate, triphosphate, polyphosphate or a        nucleic acid;    -   R⁵ is a detectable label or anchor moiety;    -   R⁷ is hydrogen or —OR^(7A), wherein R^(7A) is hydrogen;    -   R^(8A), R^(8B), R⁹, R¹⁰ and R¹¹ are as described in claim 6, and    -   X³, X⁴, X⁵, X⁶ and X⁷ are independently halogen.

9. The cleavable moiety in L³ comprises dithiol, allyl, azido,nitrobcnzyl, cyanocthyl, dimethylketal, Dde or azo.

10. The label on the base and the blocking group on the 3′—OH arechemically cleaved with high efficiency.

11. The label on the base and the blocking group on the 3′—OH aresimultaneously cleaved.

12. The label on the base and the blocking group on the 3′—OH arecleaved in separate chemical reactions.

13. Treatment of a disulfide-based linker or disulfide-based blockinggroup with a reducing agent cleaves the disulfide bond without leavingany sulthydryl remnant attached to the nucleotide added to the primer.

14. The reducing agent is THP or TCEP.

15. The polymerase is a variant of 9° N DNA polymerase or other mutatedFamily B or Family A polymerases or mutants thereof, and the reactionbuffer may contain Mn²⁺ or other divalent cations which can be used toefficiently incorporate the labeled nucleotide analogue.

An embodiment of the present invention includes a 4-color method forsequencing a nucleic acid comprising:

a) providing

-   -   1) a nucleic acid    -   2) a nucleic acid polymerase    -   3) a primer capable of hybridizing to said nucleic acid, and    -   4) four different nucleotide analogues, each comprising (i) a        base, (ii) a deoxyribose or ribose, (iii) an alkyldithiomethyl        moiety or variant thereof bound to the 3′-oxygen of the        deoxyribose or ribose including but not limited to those in        Embodiments as described herein, hereinafter referred to as a        SS-cleavable blocking group, and (iv) an anchor bound to the        base via a dithiomethyl linker or variant thereof including but        not limited to those in Embodiments described herein,        hereinafter referred to as a SS-cleavable anchors, and wherein        each nucleotide analogue comprises a unique base and a unique        anchor (including but not limited to TCO, PBA, Biotin, and        Azido);

b) incorporating with said nucleic acid polymerase one of saidnucleotide analogues into said primer to create an extended strand;

c) incubating with a correspondingly matched dye labeled bindingmolecule (including but not limited to Rox-labeled Tetrazine, Alexa-488labeled SHA, Cy5-labeled Streptavidin, and R6G-labeledDibenzocyclooctyne (DBCO)) to label the DNA products carrying the uniqueanchors on the base of the incorporated nucleotide;

d) detecting said unique detectable label of each incorporatednucleotide analogue, so as to thereby identify each incorporatednucleotide analogue in said extension strand;

e) treating said extended primer with TCEP or THP to remove theSS-blocking group and cleave the SS-cleavable linker; and

f) repeating steps a) through e) up to 30, or up to 100, or up to 1000times to determine additional nucleotide analogues added to extendedprimer strand, thereby sequencing the nucleic acid.

In an embodiment four different unlabeled nucleotide analoguesconsisting of: (i) a base, (ii) a deoxyribose or ribose, and (iii) a3′-O—S—S cleavable blocking group, hereinafter referred to as 3′—SS-NRTsare added in step a).

In an embodiment=four 3′—SS-NRTs are added with polymerase in a chasestep immediately following step a).

1-color, 4 same color labeled nucs, step by step, no chase (FIGS. 4A-4D)

Embodiment A2. A1-color method for sequencing DNA comprising:

a) providing

-   -   1) a nucleic acid    -   2) a nucleic acid polymerase    -   3) a primer capable of hybridizing to said nucleic acid, and    -   4) a labeled nucleotide analogue, comprising (i) a base, (ii) a        deoxyribose or ribose, (iii) a SS-cleavable blocking group,        and (iv) a detectable label bound to the base via a SS-cleavable        linker;

b) detecting said detectable label if the labeled nucleotide analoguehas been incorporated into the DNA primer, so as to identify theincorporated labeled nucleotide analogue in said extension strand;

c) repeating steps a) and b) with an identical labeled nucleotideanalogue except containing a different base than in a);

d) repeating steps a) and b) with an identical labeled nucleotideanalogue, except containing a different base than in a) and b);

e) repeating steps a) and b) with an identical labeled nucleotideanalogue, except containing a different base than in a), b) and c);

f) treating said extended primer with TCEP or THP to remove theSS-blocking group and cleave the SS-cleavable linker; and

g) repeating steps a) through e) up to 30, or up to 100, or up to 1000times to determine additional nucleotide analogues added to extendedprimer strand, thereby sequencing the nucleic acid.

1-color, 4 same color labeled nucs, step by step, co-chase during eachaddition (FIGS. 4A-4D)

In an embodiment of A2, the method of embodiment JS4 in which an3′—SS-NRT with the same base as the labeled nucleotide analogue is addedin steps a)4), b)4), c)4), and d)4).

1-color, 4 same color labeled nucs, step by step, post-chase after eachaddition (FIGS. 4A-4D)

In another embodiment of A2, the method of embodiment JS4 in which an3′—SS-NRT with the same base as the labeled nucleotide analogue is addedin a chase step immediately following steps a)4), b)4), c)4), and d)4).

2-color, 2 labeled nucs, 2 anchor nucs, same two labels, no chase (seeFIGS. 14A-14B)

Embodiment A3. A 2-color method for sequencing a nucleic acidcomprising:

a) providing

-   -   1) a nucleic acid    -   2) a nucleic acid polymerase    -   3) a primer capable of hybridizing to said nucleic acid,    -   4) two labeled nucleotide analogues, including but not limited        to those in Embodiments described herein comprising (i) a        base, (ii) a deoxyribose or ribose, (iii) a SS-cleavable        blocking group, and (iv) a detectable label bound to the base        via a SS-cleavable linker, in which the two labeled nucleotide        analogues bear a unique base and label (e.g.,        3′-O—SS-dATP-SS-Rox and 3′-O—SS-dCTP-SS-Alexa-488, and    -   5) two nucleotide analogues, including but not limited to those        in Embodiments described herein comprising (i) a base, (ii) a        deoxyribose or ribose, (iii) a SS-cleavable blocking group,        and (iv) an anchor moiety bound to the base via a SS-cleavable        linker, in which the two labeled nucleotides bear unique bases        different from those in 4) and two different anchors (e.g.,        3′-O—SS-dUTP-SS-N₃ and 3′-O—SS-dGTP-SS-TCO);

b) detecting said detectable label if the labeled nucleotide analoguehas been incorporated into the DNA primer, so as to identify theincorporated labeled nucleotide analogue in said extension strand, e.g.,A if Rox and C if Alexa-488;

c) incubating with a correspondingly matched dye labeled bindingmolecule including but not limited to those in Embodiments describedherein (e.g., Alexa-488-labeled Dibenzyzocyclooctyne and Rox-labeledTetrazine);

d) detecting said detectable label if the anchor nucleotide analogue hasbeen incorporated into the DNA primer, so as to identify theincorporated anchor nucleotide analogue in said extension strand, e.g.,U if Alexa-488 and G if Rox.

e) treating said extended primer with TCEP or THP to remove theSS-blocking group and cleave the SS-cleavable linker; and

f) repeating steps a) through e) up to 30, or up to 100, or up to 1000times to determine additional nucleotide analogues added to extendedprimer strand, thereby sequencing the nucleic acid.

2-color, 2 labeled nucs, 2 anchor nucs, same two labels, co-chase (seeFIGS. 14A-14B)

In an embodiment of A3, the method of embodiment JS7 in which four3′—SS-NRTs arc added in step a).

2-color, 2 labeled nues, 2 anchor nucs, same two labels, post-chase (seeFIGS. 14A-14B)

In another embodiment of A3, the method of embodiment JS4 in which four3′—SS-NRTs are added immediately after step a).

1-color, 3 anchor flues, 3 same labels (3 orthogonal cleavable linkers),1 dark NRT (see FIGS. 17A-17B)

Embodiment A4. A 1-color method for sequencing a nucleic acidcomprising:

a) providing

-   -   1) a nucleic acid    -   2) a nucleic acid polymerase    -   3) a primer capable of hybridizing to said nucleic acid, and    -   4) three anchor nucleotide analogues, including but not limited        to those in Embodiments described herein comprising (i) a        base, (ii) a deoxyribose or ribose, (iii) a SS-cleavable        blocking group, and (iv) an anchor bound to the base via a        SS-cleavable linker, in which the three labeled nucleotide        analogues each bears a unique base and anchor (e.g.,        3′-O—SS-dATP-SS—Ni, 3′-O—SS-dUTP-SS-TCO and 3′-O—        SS-dCTP-SS-Biotin, and    -   5) a 3′—SS-NRT, with a different base from those in 4) (e.g.,        3′—SS-dGTP);

b) incubating with a correspondingly matched dye labeled bindingmolecule including but not limited to those in Embodiments describedherein (e.g., DBCO-Azo Linker-ATTO647N, Tetrazine-Dde -ATTO647N andStreptavidin-ATTO647N) to label the DNA products carrying the uniqueanchors on the base of the incorporated nucleotide;

c) detecting said detectable label if the anchor nucleotide analogue hasbeen incorporated into the DNA primer, so as to identify theincorporated anchor nucleotide analogue in said extension strand (e.g.,A, C or T incorporated if fluorescent, G if not);

d) treating said extended primer with sodium dithionite to cleave theazo linkage if such nucleotide has been incorporated;

e) detecting said detectable label (e.g., if label lost, indicates A wasincorporated);

f) treating said extended primer with hydrazine to cleave the Ddelinkage if such nucleotide has been incorporated;

g) detecting said detectable label (e.g., if label now lost, indicates Uwas incorporated);

h) treating said extended primer with TCEP or THP to cleave the S—S bondwhich would remove any remaining dye and reinstall a 3′—OH at the sametime;

i) detecting said detectable label (e.g., if label now lost, indicates Cwas incorporated);

j) optionally, chasing with three 3′—SS-dNTPs not already used;

k) repeating steps a) through j) up to 30, or up to 100, or up to 1000times to determine additional nucleotide analogues added to extendedprimer strand; thereby sequencing the nucleic acid.

1-color, 2 anchor nucs, 2 same color labels (cleavable), 1 same colorlabeled nue, 1 dark NRT (see FIGS. 19A-19B)

Embodiment A5. A 1-color method for sequencing a nucleic acidcomprising:

a) providing

-   -   1) a nucleic acid    -   2) a nucleic acid polymerase    -   3) a primer capable of hybridizing to said nucleic acid,    -   4) two anchor nucleotide analogues, including but not limited to        those in Embodiments described herein, comprising (i) a        base, (ii) a deoxyribose or ribose, (iii) a SS-cleavable        blocking group, and (iv) an anchor bound to the base via a        SS-cleavable linker, in which the two labeled nucleotide        analogues each bears a unique base and anchor (e.g.,        3′-O—SS-dUTP-SS—N₃ and 3′-O—SS-dUTP-SS-Biotin, and    -   5) one labeled nucleotide analogue, including but not limited to        those in Embodiments described herein with a different base from        those in 4) (e.g., 3′-O—SS-dATP-Rox);

6) one 3′—SS-NRT, with a base different from those in 4) and 5);

b) detecting said detectable label if the anchor nucleotide analogue hasbeen incorporated into the DNA primer, so as to identify theincorporated anchor nucleotide analogue in said extension strand (e.g.,A if fluorescent, C, G or T if not);

c) incubating with two correspondingly matched dye labeled bindingmolecules including but not limited to those in Embodiments describedherein (e.g., Rox-labeled DBCO-Azo Linker and Rox-labeled Streptavidin)to label the DNA products carrying the unique anchors on the base of theincorporated nucleotide;

d) detecting said detectable label if the anchor nucleotide analogue hasbeen incorporated into the DNA primer, so as to identify theincorporated anchor nucleotide analogue in said extension strand (e.g.,A, C or T if fluorescent, G if not);

d) treating said extended primer with sodium dithionite to cleave theazo linkage if such nucleotide has been incorporated;

e) detecting said detectable label (e.g., if label lost, indicates T wasincorporated);

f) treating said extended primer with TCEP or THP to cleave the S—S bondwhich would remove any remaining dye and reinstall a 3′—OH at the sametime;

g) detecting said detectable label (e.g., if label now lost, indicates Cwas incorporated, since if A, would have been determined in step b);

h) optionally, chasing with four 3′—SS-dNTPs used;

i) repeating steps a) through h) up to 30, or up to 100, or up to 1000times to determine additional nucleotide analogues added to extendedprimer strand;

thereby sequencing the nucleic acid.

1-color, 3 anchor nucs, 3 same dye labels (orthogonally cleavablelinkers), 1 labeled nuc (see FIGS. 21A-21F)

Embodiment A6. A 1-color method for sequencing a nucleic acidcomprising:

a) providing

-   -   1) a nucleic acid    -   2) a nucleic acid polymerase    -   3) a primer capable of hybridizing to said nucleic acid,    -   4) three anchor nucleotide analogues, including but not limited        to those in Embodiments described herein comprising (i) a        base, (ii) a deoxyribose or ribose, (iii) a SS-cleavable        blocking group, and (iv) an anchor bound to the base via a        SS-cleavable linker, in which the two labeled nucleotide        analogues each bears a unique base and anchor (e.g.,        3′-O—SS-dGTP-SS-N3, 3′-O—SS-dCTP-Biotin and 3′-O—SS-dATP-TCO,        and    -   5) one labeled nucleotide analogue, including but not limited to        those in Embodiments described herein with a different base from        those in 4) (e.g., 3′-O—SS-dATP-SS-Rox);

b) detecting said detectable label if the anchor nucleotide analogue hasbeen incorporated into the DNA primer, so as to identify theincorporated anchor nucleotide analogue in said extension strand (e.g.,A if fluorescent, C, G or T if not);

c) incubating with correspondingly matched dye labeled binding moleculesincluding but not limited to those in Embodiments described herein(e.g., Rox-Azo-labeled DBCO, Rox-labeled Tetrazine-Azo-Linker andRox-labeled Streptavidin) to label the DNA products carrying the uniqueanchors on the base of the incorporated nucleotide;

d) detecting said detectable label if the anchor nucleotide analogue hasbeen incorporated into the DNA primer, so as to identify theincorporated anchor nucleotide analogue in said extension strand (e.g.,C, G or T if now fluorescent);

d) treating said extended primer with sodium dithionite to cleave theazo linkage if such nucleotide has been incorporated;

e) detecting said detectable label (e.g., if label lost, indicates G wasincorporated);

f) treating said extended primer with hydrazine to cleave the Ddelinkage if such nucleotide has been incorporated;

g) detecting said detectable label (e.g., if label now lost, indicates Uwas incorporated, otherwise C);

h) treating said extended primer with TCEP or THP to cleave the S—S bondwhich would remove any remaining dye and reinstall a 3′—OH at the sametime;

i) detecting said detectable label (e.g., if label now lost, confirms Cwas incorporated, since if A, would have been determined in step b));

j) optionally, chasing with four 3′—SS-dNTPs;

k) repeating steps a) through h) up to 30, or up to 100, or up to 1000times to determine additional nucleotide analogues added to extendedprimer strand; thereby sequencing the nucleic acid.

1-color, 3 labeled nucs, 2 same color labels (orthogonally cleavablelinkers), 1 dark NRT (see FIGS. 23A-23D)

Embodiment A7. A1-color method for sequencing a nucleic acid comprising:

a) providing

-   -   1) a nucleic acid    -   2) a nucleic acid polymerase    -   3) a primer capable of hybridizing to said nucleic acid,    -   4) three labeled nucleotide analogues, including but not limited        to those in Embodiments described herein comprising (i) a        base, (ii) a deoxyribose or ribose, (iii) a SS-cleavable        blocking group, and (iv) a label bound to the base via a        chemically or photocleavable linker, in which the three labeled        nucleotide analogues each bears a unique base, cleavable linker        and label (e.g., 3′-O—SS-dATP-SS-Rox, 3′-O—SS-dUTP-Allyl-Rox and        3′-O—SS-dCTP-Nitrobenzyl-Rox, and    -   5) one 3′—SS-NRT with a different base from those in 4) (e.g.,        3′-O-t-Butyl-SS-dGTP);

b) detecting said detectable label if the labeled nucleotide analoguehas been incorporated into the DNA primer, so as to identify theincorporated anchor nucleotide analogue in said extension strand (e.g.,A, U or C if fluorescent, G if not);

c) photo-irradiating at ˜350 nm to cleave the nitrobenzyl-containinglinkers if such nucleotide has been incorporated;

d) detecting said detectable label if the labeled nucleotide analoguehas been incorporated into the DNA primer, so as to identify theincorporated anchor nucleotide analogue in said extension strand (e.g.,if label lost, C was incorporated);

e) cleavage with Pd (O) to cleave the allyl-containing linkers if suchnucleotide has been incorporated;

f) detecting said detectable label if the labeled nucleotide analoguehas been incorporated into the DNA primer, so as to identify theincorporated anchor nucleotide analogue in said extension strand (e.g.,if label lost, U was incorporated);

g) treating said extended primer with TCEP or THP to cleave the S—S bondwhich would remove any remaining dye and reinstall a 3′—OH at the sametime;

h) detecting said detectable label (e.g., if label now lost, confirms Awas incorporated);

j) optionally, chasing with three 3′-SS-dNTPs not already used;

k) repeating steps a) through h) up to 30, or up to 100, or up to 1000times to determine additional nucleotide analogues added to extendedprimer strand; thereby sequencing the nucleic acid.

4-color, 4 labeled nucs, no chase (see FIGS. 26A-26F)

Embodiment A8.A4-color method for sequencing a nucleic acid comprising:

a) providing

-   -   1) a nucleic acid    -   2) a nucleic acid polymerase    -   3) a primer capable of hybridizing to said nucleic acid, and    -   4) four different nucleotide analogues, each comprising (i) a        base, (ii) a deoxyribose or ribose, (iii) an alkyldithiomethyl        moiety or variant thereof bound to the 3′-oxygen of the        deoxyribose or ribose including but not limited to those in        Embodiments as described herein hereinafter referred to as a        SS-cleavable blocking group, and (iv) a label bound to the base        via a dithiomethyl linker or variant thereof including but not        limited to those in Embodiments described herein, hereinafter        referred to as a SS-cleavable anchors, and wherein each        nucleotide analogue comprises a unique base and a unique label        (including but not limited to Rox, Alexa-488, Cy5 and R6G)        (e.g., 3″-O—SS-dATP-SS-Rox, 3′-O—SS-dCTP-SS-Alexa-488,        3′-O—SS-dGTP-SS-Cy5, and 3′-O—SS-dUTP-R6G)

b) incorporating with said nucleic acid polymerase one of saidnucleotide analogues into said primer to create an extended strand;

c) detecting said unique detectable label of each incorporatednucleotide analogue, so as to thereby identify each incorporatednucleotide analogue in said extension strand;

d) treating said extended primer with TCEP or THP to remove theSS-blocking group and cleave the SS-cleavable linker; and

e) repeating steps a) through d) up to 30, or up to 100, or up to 1000times to determine additional nucleotide analogues added to extendedprimer strand, thereby sequencing the nucleic acid.

4-color, 4 labeled nucs, mix label and unlabel (see FIGS. 27A-27B)

In an embodiment of A8, the method of embodiment JS14 in which all four3′—SS-NRTs are added in step a).

4-color, 4 labeled nucs, chase (see FIGS. 25A-25F)

In another embodiment of A8, the method of embodiment JS14 in which allfour 3′—SS-NRTs are added in a chase step immediately following step a).[0372] General sequencing plus walking (see FIG. 28 )

Embodiment A9. A4-color method for sequencing and walking within anucleic acid comprising:

a) obtaining a sequence of up to 100 or up to 200 nucleotides using anyof the methods of Claims S1 to S14 or other sequencing methods known inthe art.

b) denaturing the DNA to strip off the extended primer and reannealingthe original primer;

c) adding a mixture containing three natural nucleotides (e.g., dATP,dCTP and dTTP) and one 3′-O—SS-dNTP (e.g., 3′-O—SS-dGTP) (“walkingstep”) to extend the primer in a single step to the next C in thetemplate;

d) adding TCEP or THP to restore the 3′—OH group on the lastincorporated nucleotide;

e) repeating steps c) and d) enough times to reach approximately theposition where the original sequencing run ended;

f) repeating steps a) to e) to sequence another stretch of the nucleicacid and walk to the position where the second sequencing run ended;

g) repeating step j) as necessary to obtain long assembled read (3 or 4times the length of any individual read), thereby obtaining a longstretch of nucleic acid synthesis.

4-color sequencing plus walking (see FIG. 28A-28B)

Embodiment A10.A4-color method for sequencing and walking within anucleic acid comprising:

a) providing

-   -   1) a nucleic acid    -   2) a nucleic acid polymerase    -   3) a primer capable of hybridizing to said nucleic acid, and    -   4) four different nucleotide analogues, each comprising (i) a        base, (ii) a deoxyribose or ribose, (iii) an alkyldithiomethyl        moiety or variant thereof bound to the 3′-oxygen of the        deoxyribose or ribose including but not limited to those in        Embodiments described herein, hereinafter referred to as a        SS-cleavable blocking group, and (iv) a label bound to the base        via a dithiomethyl linker or variant thereof including but not        limited to those in Embodiments described herein, hereinafter        referred to as a SS-cleavable anchors, and wherein each        nucleotide analogue comprises a unique base and a unique label        (including but not limited to Rox, Alexa-488, Cy5 and R6G)        (e.g., 3′-O—SS-dATP-SS-Rox, 3′-O—SS-dCTP-SS-Alexa-488,        3′-O—SS-dGTP-SS-Cy5, and 3′-O—SS-dUTP-R6G)

b) incorporating with said nucleic acid polymerase one of saidnucleotide analogues into said primer to create an extended strand;

c) detecting said unique detectable label of each incorporatednucleotide analogue, so as to thereby identify each incorporatednucleotide analogue in said extension strand;

d) treating said extended primer with TCEP or THP to remove theSS-blocking group and cleave the SS-cleavable linker; and

e) repeating steps a) through d) up to 100 times to determine additionalnucleotide analogues added to extended primer strand,

f) denaturing the DNA to strip off the extended primer and rcannealingthe original primer;

g) adding a mixture containing three natural nucleotides (e.g., dATP,dCTP and dTTP) and one 3′-O—SS-dNTP (e.g., 3′-O—SS-dGTP) (“walkingstep”) to extend the primer in a single step to the next C in thetemplate;

h) adding TCEP or THP to restore the 3′—OH group on the lastincorporated nucleotide;

i) repeating steps g) and h) enough times to reach approximately theposition where the original sequencing run ended;

j) repeating steps a) to i) to sequence another stretch of the nucleicacid and walk to the position where the second sequencing run ended;

k) repeating step j) as necessary to obtain long assembled read (3 or 4times the length of any individual read),

-   -   thereby obtaining a long stretch of nucleic acid synthesis.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

Embodiments

While various embodiments of the invention are shown and describedherein, it will be obvious to those skilled in the art that suchembodiments arc provided by way of example only. Numerous variations,changes, and substitutes may occur to those skilled in the art withoutdeparting from the invention. It should be understood that variousalternatives to the embodiments of the invention described herein may beemployed.

As used herein, and unless stated otherwise, each of the following termsshall have the definition set forth below:

-   A Adenine;-   C Cytosine;-   DNA Deoxyribonucleic acid;-   G Guanine;-   RNA Ribonucleic acid;-   T Thyminc; and-   U Uracil.

The articles “a”, “an” and “the” are non-limiting. For example, “themethod” includes the broadest definition of the meaning of the phrase,which can be more than one method.

“Nucleic acid” shall mean any nucleic acid molecule and its derivatives,including, without limitation, DNA, RNA and hybrids thereof The nucleicacid bases that form nucleic acid molecules can be the bases A, C, G, Tand U, as well as derivatives thereof. Derivatives of these bases arewell known in the art, and are exemplified in PCR Systems, Reagents andConsumables (Perkin Elmer Catalogue 1996-1997, Roche Molecular Systems,Inc., Branchburg, N.J., USA).

As used herein, “nucleotide analogue” shall mean an analogue of A, G, C,T or U (that is, an analogue of a nucleotide comprising the base A, G.C, T or U), comprising a phosphate group, which is recognized by DNA orRNA polymerase (whichever is applicable) and incorporated into a strandof DNA or RNA (whichever is appropriate). Examples of nucleotideanalogues include, without limitation, 7-deaza-adenine, 7-deaza-guanine,the analogues of deoxynucleotides shown in herein analogues in which alabel is attached through a cleavable linker to the 5-position ofcytosine or thymine or to the 7-position of deaza-adenine ordeaza-guanine, and analogues in which a small chemical moiety is used tocap the —OH group at the 3′-position of deoxyribose. Nucleotideanalogues and DNA polymerase-based DNA sequencing are also described inU.S. Pat. No. 6,664,079.

All embodiments of U.S. Pat. No. 6,664,079 (the contents of which arehereby incorporated by reference) with regard to sequencing a nucleicacid are specifically envisioned here.

“Alkyldithiomethyl” refers to a compound, or portion thereof, comprisinga dithio group, where one of the sulfurs is directly connected to amethyl group and the other sulfur is directly connected to an alkylgroup. An example is the structure

wherein R is an alkyl group and the wavy line represents a point ofconnection to another portion of the compound. In some cases, thealkyldithiomethyl is methyldithiomethyl, ethyldithiomethyl,propyldithiomethyl, isopropyldithiomethyl, butyldithiomethyl,t-butyldithiomethyl_(—) or phenyldithiomethyl.

Embodiment P1. A nucleotide analogue comprising (i) a base, (ii) adeoxyribose or ribose, (iii) an alkyldithiomethyl moiety bound to the3′-oxygen of the deoxyribose or ribose, and (iv) a detectable labelbound to the base via a dithiomethyl linker.

Embodiment P2. The nucleotide analogue of embodiment P1, wherein thenucleotide analogue comprises a deoxyribose.

Embodiment P3. The nucleotide analogue of embodiments P1, wherein thenucleotide analogue comprises a ribose.

Embodiment P4. The nucleotide analogue of any of embodiments P1-3,wherein the nucleotide analogue is a nucleoside triphosphate, anucleoside tetraphosphate, a nucleoside pentaphosphate, or a nucleosidehexaphosphate.

Embodiment P5. The nucleotide analogue of any of embodiments P1-4,wherein the base is selected from the group consisting of adenine or ananalogue of adenine, guanine or an analogue of guanine, cytosine or ananalogue of cytosine, thymine or an analogue of thymine and uracil or ananalogue of uracil.

Embodiment P6. The nucleotide analogue of any of embodiments P1-5,wherein the alkyldithiomethyl moiety bound to the 3′-oxygen is selectedfrom the group consisting of methyldithiomethyl, ethyldithiomethyl,propyldithiomethyl, isopropyldithiomethyl, butyldithiomethyl,t-butyldithiomethyl, and phenyldithiomethyl.

Embodiment P7. The nucleotide analogue of any of embodiments P1-6,wherein the alkyldithiomethyl moiety has the structure

wherein R is the alkyl portion of the alkyldithiomethyl moiety and thewavy line represents the point of connection to the 3′-oxygen.

Embodiment P8. The nucleotide analogue of any of embodiments P1-7,wherein the detectable label bound to the base via a dithiomethyl linkeris bound to the 5-position of the base if the base is T, U, or C or ananalogue of T, U, or C, and to the 7-position of the base if the base isA or G or an analogue of A or G.

Embodiment P9. The nucleotide analogue of any of embodiments P1-8,wherein the base is a deaza analogue.

Embodiment P10. The nucleotide analogue of embodiment P9, wherein thedeaza analogue is a 7-deazapurine.

Embodiment P11. The nucleotide analogue of any of embodiments P1-10,wherein the alkyldithiomethyl moiety and the dithiomethyl linker arcboth cleavable with tris-(2-carboxyethyl)phosphine (TCEP) ortris(hydroxypropyl)phosphine (THP).

Embodiment P12. The nucleotide analogue of any of embodiments P1-11,wherein the dithiomethyl linker has a structure as follows:

wherein α represents one or more atoms through which a covalentconnection is established to the base, and β represents one or moreatoms through which a covalent connection is established to thedetectable label.

Embodiment P13. The nucleotide analogue of embodiment P12, wherein thedithiomethyl linker has a structure as follows:

wherein α′ represents one or more atoms through which a covalentconnection is established to the base, and β′ represents one or moreatoms through which a covalent connection is established to thedetectable label.

Embodiment P14. The nucleotide analogue of embodiments P13, wherein thedithiomethyl linker is included within a a structure as follows:

wherein B represents the point of connection to the base; wherein Lrepresents the point of connection to the detectable label; and whereinn is 1-11.

Embodiment P15. The nucleotide analogue of any of embodiments P1-14,wherein the detectable label is selected from the group consisting of adye, a fluorophore, a combinatorial fluorescence energy transfer tag, achemiluminescent compound, a chromophore, a mass tag, and an el ectrophore.

Embodiment P16. The nucleotide analogue of embodiments P15, wherein thedetectable label is a fluorophore.

Embodiment P17. The nucleotide analogue of embodiments P16, wherein thefluorophore is selected from the group including but not limited toBodipyFL, R6G, ROX, and Cy5.

Embodiment P18. The nucleotide analogue of embodiments P1, wherein thenucleotide analogue is selected from the group consisting of3′-O-t-butyl-dithiomethyl-dCTP—S—S-BodipyFL,3′-O-t-butyl-dithiomethyl-dUTP—S—S-R6G,3′-O-t-butyl-dithiomethyl-dATP—S—S-ROX, and3′-O-t-butyl-dithiomethyl-dGTP—S—S-Cy5, where S-S represents the dithiolinker.

Embodiment P19. The nucleotide analogue of embodiments P 1, wherein thestructure of the nucleotide analogue is selected from

wherein R is methyl, ethyl, propyl, isopropyl, butyl, t-butyl, orphenyl; n is 2-11, and m is 1-4.

Embodiment P20. A composition comprising at least two different types ofa nucleotide analogue of any of embodiments P1-18, wherein each type ofnucleotide analogue comprises a different base and a differentdetectable label from each of the other types of nucleotide analogue.

Embodiment P21. A composition comprising a first type of nucleotideanalogue of any of claims 1-18 and a second type of nucleotide analogueof any of embodiments P1-18, wherein the second type of nucleotideanalogue comprises a different base and a different detectable labelfrom the first type of nucleotide analogue.

Embodiment P22. The composition of embodiments P21, further comprising athird type of nucleotide analogue of any of claims 1-18, wherein thethird type of nucleotide analogue comprises a different base and adifferent detectable label from each of the other two types ofnucleotide analogue.

Embodiment P23. The composition of embodiments P22, further comprising afourth type of nucleotide analogue of any of claims 1-18, wherein thefourth type of nucleotide analogue comprises a different base and adifferent detectable label from each of the other three types ofnucleotide analogue.

Embodiment P24. The composition of embodiments P23, further comprising afifth type of nucleotide analogue of any of claims 1-18, wherein thefifth type of nucleotide analogue comprises a different base and adifferent detectable label from each of the other four types ofnucleotide analogue.

Embodiment P25. A nucleotide analogue comprising (i) a base selectedfrom the group consisting of adenine or an analogue of adenine, guanineor an analogue of guanine, cytosine or an analogue of cytosine, thymineor an analogue of thymine and uracil or an analogue of uracil, (ii) adeoxyribose or ribose, (iii) an alkyldithiomethyl moiety bound to the3′-oxygen of the deoxyribose or ribose, and (iv) a 3-aminopropynyl groupbound to the 5-position of the base if the base is T, U, or C or ananalogue of T, U, or C, and bound to the 7-position of the base if thebase is A or G or an analogue of A or G.

Embodiment P26. A method for sequencing a nucleic acid, comprising:

a) providing

-   -   1. a nucleic acid,    -   2. a nucleic acid polymerase,    -   3. a primer capable of hybridizing to said nucleic acid, and    -   4. four different labeled nucleotide analogues, each        comprising (i) a base, (ii) a deoxyribose or ribose, (iii) an        alkyldithiomethyl moiety bound to the 3′-oxygen of the        deoxyribose or ribose, and (iv) a detectable label bound to the        base via a dithiomethyl linker, and wherein each nucleotide        analogue comprises a unique base and a unique detectable label;

b) incorporating with said nucleic acid polymerase one or more of saidnucleotide analogues into said primer to create an extension strand;

c) detecting said unique detectable label of each incorporatednucleotide analogue, so as to thereby identify each incorporatednucleotide analogue in said extension strand, thereby sequencing thenucleic acid.

Embodiment P27. A method for sequencing a nucleic acid, comprising:

a) providing

a) a nucleic acid,

b) a nucleic acid polymerase,

c) a primer capable of hybridizing to said nucleic acid,

d) three different types of labeled nucleotide analogues, eachcomprising (i) a base, (ii) a deoxyribose or ribose, (iii) analkyldithiomethyl moiety bound to the 3′-oxygen of the deoxyribose orribose, and (iv) a detectable label bound to the base via a dithiomethyllinker, and wherein each nucleotide analogue comprises a unique base anda unique detectable label; and

e) an unlabeled nucleotide analogue, comprising (i) a base, (ii) adeoxyribose or ribose, and (iii) an alkyldithiomethyl moiety bound tothe 3′-oxygen of the deoxyribose or ribose, and wherein the base isdifferent from each base of the labeled nucleotide analogues;

b) incorporating with said nucleic acid polymerase one or more of saidnucleotide analogues into said primer to create an extension strand;

c) detecting a unique detectable label, if present, of each incorporatednucleotide analogue, so as to thereby identify each incorporatednucleotide analogue in said extension strand,

thereby sequencing the nucleic acid.

Embodiment P28. The method of any of embodiments P26-27, furthercomprising removing the alkyldithiomethyl moiety bound to the 3′-oxygenof the deoxyribose or ribose by cleaving the S—S bond, so as to permitincorporation of another analogue into each of said extension strands.

Embodiment P29. The method of any of embodiments P26-28, furthercomprising removing a unique detectable label, if present, from eachincorporated nucleotide analogue by cleaving the dithio bond.

Embodiment P30. The method of any of embodiments P28-29, wherein thedithio bond in at least one of the alkyldithiomethyl moiety and thedithiomethyl linker, if present, is cleaved bytris-(2-carboxyethyl)phosphine (TCEP) or tris(hydroxypropyl)phosphine(THP).

Embodiment P31. The method of any of embodiments P26-30, wherein eachnucleoside analogue is a nucleoside triphosphate, a nucleosidetetraphosphate, a nucleoside pentaphosphate, or a nucleosidehexaphosphate.

Embodiment P32. The method of any of embodiments P26-31, wherein eachbase is selected from the group consisting of adenine or an analogue ofadenine, guanine or an analogue of guanine, cytosine or an analogue ofcytosine, thymine or an analogue of thymine and uracil or an analogue ofuracil.

Embodiment P33. The method of any of embodiments P26-32, wherein thenucleotide analogue comprises a deoxyribose.

Embodiment P34. The method of embodiments P33, wherein the polymerase isa DNA polymerase and the nucleic acid is DNA.

Embodiment P35. The method of embodiments P33, wherein the polymerase isa reverse transcriptase and the nucleic acid is RNA.

Embodiment P36. The method of any of embodiments P26-32, wherein thenucleotide analogue comprises a ribose.

Embodiment P37. The method of embodiments P36, wherein the polymerase isa DNA-based RNA polymerase and the nucleic acid is DNA.

Embodiment P38. The method of embodiments P36, wherein the polymerase isan RNA-based RNA polymerase and the nucleic acid is RNA.

Embodiment P39. The method of any of embodiments P26-38, wherein eachalkyldithiomethyl moiety bound to the 3′-oxygen is independentlyselected from the group consisting of methyldithiomethyl,ethyldithiomethyl, propyldithiomethyl, isopropyldithiomethyl,butyldithiomethyl, t-butyldithiomethyl, and phenyldithiomethyl.

Embodiment P40. The method of any of embodiments P25-39, wherein eachalkyldithiomethyl moiety has the structure

wherein R is the alkyl portion of the alkyldithiomethyl moiety and thewavy line represents the point of connection to the 3′-oxygen.

Embodiment P41. The method of any of embodiments P26-40, wherein eachdetectable label bound to the base via a dithiomethyl linker is bound tothe 5-position of the base if the base is T, U, or C or an analogue ofT, U, or C, and to the 7-position of the base if the base is A or G oran analogue of A or G.

Embodiment P42. The method of any of embodiments P26-41, wherein thebase of at least one of the nucleotide analogues is a dcaza analogue.

Embodiment P43. The method of embodiments P42, wherein the deazaanalogue is a 7-deazapurine.

Embodiment P44. The method of any of embodiments P25-43, wherein eachdithiomethyl linker has a structure as follows:

wherein α represents one or more atoms through which a covalentconnection is established to the base, and β represents one or moreatoms through which a covalent connection is established to thedetectable label.

Embodiment P45. The method of embodiment P44, wherein each dithiomethyllinker has a structure as follows:

wherein α′ represents one or more atoms through which a covalentconnection is established to the base, and β′ represents one or moreatoms through which a covalent connection is established to thedetectable label.

Embodiment P46. The method of embodiment P45, wherein each dithiomethyllinker is included within a structure as follows:

wherein B represents the point of connection to the base; wherein Lrepresents the point of connection to the detectable label; and whereinn is 1-11.

Embodiment P47. The method of any of embodiments P26-46, wherein eachdetectable label is selected from the group consisting of a dye, afluorophore, a combinatorial fluorescence energy transfer tag. achemiluminescent compound, a chromophore, a mass tag, and anelectrophore.

Embodiment P48. The method of embodiments P47, wherein each detectablelabel is a fluorophore.

Embodiment P49. The method of embodiment P48, wherein the fluorophore isselected from the group consisting of BodipyFL, R6G, ROX, and Cy5.

Embodiment P50. The method of any of embodiments P26-27, wherein eachlabeled nucleotide analogue is selected from the group consisting of3′-O-t-butyl-dithiomethyl-dCTP—S—S-BodipyFL,3′-O-t-butyl-dithiomethyl-dUTP—S—S-R6G,3′-O-t-butyl-dithiomethyl-dATP—S—S-ROX, and3′-O-t-butyl-dithiomethyl-dGTP—S—S-Cy5, where S-S represents a dithiolinker.

Embodiment P51. The method of any of embodiments P26-27, wherein thestructure of each labeled nucleotide analogue is selected from

wherein R is methyl, ethyl, propyl, isopropyl, butyl, t-butyl, orphenyl; n is 1-11, and m is 1-4.

Embodiment P52. The method of any of embodiments P26-51, wherein thenucleic acid is immobilized on a solid substrate.

Embodiment P53. The method of embodiment P52, wherein the nucleic acidis immobilized on the solid substrate via a 1,3-dipolar cycloadditionreaction between an azido and alkyne functional group, or abiotin-streptavidin interaction.

Embodiment P54. The method of any of embodiments P52-53, wherein thesolid substrate is in the form of a chip, a bead, a well, a capillarytube, or a slide.

Embodiment P55. The method of any of embodiments P52-54, wherein thesolid substrate is gold, quartz, silica, or plastic.

Embodiment P56. The method of any of embodiments P52-55, wherein thesolid substrate is porous.

Embodiment P57. The method of any of embodiments P26-56, simultaneouslyapplied to a plurality of different nucleic acids.

Embodiment P58. A process for producing a 3′-O-ethyldithiomethylnucleoside, comprising:

a) providing,

a) a nucleoside,

b) acetic acid,

c) acetic anhydride, and

d) DMSO

under conditions permitting the production of a 3′-O-methylthiomethylnucleoside;

b) contacting the 3′-O-methylthiomethyl nucleoside produced in part a)with trimethylamine, molecular sieve, and sulfuryl chloride underconditions permitting the production of a 3′-O-chloromethyl nucleoside;

c) contacting the 3′-O-chloromethyl nucleoside produced in part b) withpotassium p-toluenethiosulfonate and ethanethiol under conditionspermitting the production of a 3′-O-ethyldithiomethyl nucleoside.

Embodiment P59. A process for producing an-(3-aminopropynyl)-3′-O-t-butyldithiomethyl-dNTP, wherein n is 5 if thebase is C, T, or U, and n is 7 if the base is A or G, using the examplehere for the synthesis of 5-(3-aminopropynyl)-3′-O-t-butyl dith o methyl -dCTP, comprising:

a) providing

-   -   a) a 5-iodo-2′-deoxy nucleoside (C), and

b) N,N-dimethylformamide dimethyl acetal under conditions permitting theformation of a N⁴-DMF-5-iodo-2′-deoxy nucleoside (C); contacting theN⁴-DMF-5-iodo-2′-deoxy nucleoside produced in step a) with trityl-Clunder conditions permitting the formation of aN⁴-DMF-5-iodo-5′-O-trityl-2′-deoxy nucleoside (C);

c) contacting the N⁴-DMF-5-iodo-5′-O-trityl-2′-deoxy nucleoside (C)produced in step b) with triethylamine and N-propargyltrifluoroacetamide under conditions permitting the formation of aN⁴-DMF-5-[3-(trifluoroacetamido)propynyl]-5′-O-trityl-2,-deoxynucleoside (C);

d) contacting theN⁴-DMF-5[3-(trifluoroacetamido)propynyl]-5′-O-trityl-2′-deoxy nucleoside(C) produced in step c) with DMSO, acetic acid and acetic anhydrideunder conditions permitting the formation of aN⁴-DMF-5-[3-(trifluoroacetamido)propynyl]-5′-O-trityl-3′-O-methylthiomethyl-2′-deoxynucleoside (C);

e) contacting theN⁴-DMF-5-[3-(trifluoroacetamido)propynyl]-5′-O-trityl-3′-O-methylthiomethyl-T-deoxynucleoside (C) produced in step d) with triethylamine, molecular sieves,sulfuryl chloride, potassium p-toluenethiosulfonate, and t-butylmercaptan, under conditions permitting the formation of aN⁴-DMF-5-[3-(trifluoroacetamido)propynyl]-5′-O-trityl-3′-O-(t-butyldithiomethyl)-2′-deoxynucleoside (C);

f) contacting theN⁴-DMF-5-[3-(trifluoroacetamido)propynyl]-5′-O-trityl-3′-O-(t-butyldithiomethyl)-2′-deoxynucleoside (C) produced in step e) with trichloroacetic acid underconditions permitting the formation of aN⁴-DMF-5-[3-(trifluoroacetamido)propynyl]-3′-O-(t-butyldithiomethyl)-2′-deoxynucleoside (C);

g) contacting theN⁴-DMF-5-[3-(trifluoroacetamido)propynyl]-3′-O-(t-butyldithiomethyl)-2′-deoxynucleoside (C) produced in step f) with tetrabutylammoniumpyrophosphate, tributylamine, I₂, pyridine, and NH₄OH under conditionspermitting the formation of a5-[3aminopropynyl]-3′-O-(t-butyldithiomethyl)-dCTP.

Embodiment P60. A process for producing a3′-O-alkyldithiomethyl-dNTP-SS-dye, where SS is an alkyldithio linker,comprising:

354. providing

a) a compound comprising the structure

-   -   wherein α represents one or more atoms through which a covalent        connection is established to a carboxylic acid group, and β        represents one or more atoms through which a covalent connection        is established to a dye, and

b) a 3′-O-alkyldithiomethyl-dNTP-n-(3-aminopropynyl), wherein n is 5 ifthe base is C, T, or U, and n is 7 if the base is A or G, underconditions permitting the formation of a3′-O-alkyldithiomethyl-dNTP-SS-dye.

Embodiment P61. A plurality of different nucleic acids immobilized on asolid substrate and hybridized to primers, a portion of said primerscomprising incorporated nucleotide analogues, said nucleotide analoguescomprising (i) a base, (ii) a deoxyribose or a ribose, (iii) analkyldithiomethyl moiety bound to the 3′-oxygen of the deoxyribose orribose, and (iv) a detectable label bound to the base via a dithiomethyllinker.

Embodiment P62. The plurality of different nucleic acids of embodimentsP61, wherein each base is selected from the group consisting of adenineor an analogue of adenine, guanine or an analogue of guanine, cytosineor an analogue of cytosine, thymine or an analogue of thymine and uracilor an analogue of uracil.

Embodiment P63. The plurality of different nucleic acids of any ofembodiments P61-62, wherein said alkyldithiomethyl moieties bound to the3′-oxygen are selected from the group consisting of methyldithiomethyl,ethyldithiomethyl, propyldithiomethyl, isopropyldithiomethyl,butyldithiomethyl, t-butyldithiomethyl, and phenyldithiomethyl.

Embodiment P64. The plurality of different nucleic acids of any ofembodiments P61-63, wherein each alkyldithiomethyl moiety has thestructure

wherein R is the alkyl portion of the alkyldithiomethyl moiety and thewavy line represents the point of connection to the 3′-oxygen.

Embodiment P65. The plurality of different nucleic acids of any ofembodiments P61-64, wherein at least one of said nucleotide analogues isa deaza analogue.

Embodiment P66. The plurality of different nucleic acids of embodimentsP65, wherein the deaza analogue is a 7-deazapurine.

Embodiment P67. The plurality of different nucleic acids of any ofembodiments P61-66, wherein each linker has a structure as follows:

wherein α represents one or more atoms through which a covalentconnection is established to the base, and β represents one or moreatoms through which a covalent connection is established to thedetectable label.

Embodiment P68. The plurality of different nucleic acids of embodimentsP67, wherein each dithiomethyl linker has a structure as follows:

wherein α′ represents one or more atoms through which a covalentconnection is established to the base, and β′ represents one or moreatoms through which a covalent connection is established to thedetectable label.

Embodiment P69. The plurality of different nucleic acids of embodimentsP68, wherein each linker is included within a structure as follows:

wherein B represents the point of connection to the base; wherein Lrepresents the point of connection to the detectable label; and whereinn is 1-11.

Embodiment P70. The plurality of different nucleic acids of any ofembodiments P61-69, wherein said detectable labels are selected from thegroup consisting of a dye, a fluorophore, a combinatorial fluorescenceenergy transfer tag, a chemiluminescent compound, a chromophore, a masstag, and an electrophore.

Embodiment P71. The plurality of different nucleic acids of embodimentsP70, wherein said detectable labels are fluorophores.

Embodiment P72. A kit for nucleic acid sequencing, comprising, inseparate compartments:

a) a plurality of nucleotide analogues, each comprising (i) a base, (ii)a deoxyribose or ribose, (iii) an alkyldithiomethyl moiety bound to the3′-oxygen of the deoxyribose or ribose, and (iv) a detectable labelbound to the base via a dithiomethyl linker;

b) reagents suitable for use in nucleic acid polymerization; and

c) instructions for use.

Embodiment P73. The kit of embodiments P72, further comprising

a) a nucleotide analogue, comprising (i) a base, (ii) a deoxyribose orribose, and (iii) an alkyldithiomethyl moiety bound to the 3′-oxygen ofthe deoxyribose or ribose.

Additional Embodiments

Embodiment 1. A compound of the formula:

wherein

-   -   B is a base; L¹ is covalent linker; L² is covalent linker;    -   R³ is —OH, monophosphate, polyphosphate or a nucleic acid;    -   R^(4A) is hydrogen, —CH₃, —CX¹ ₃, —CHX¹ ₂, —CH₂X¹, —OCX¹ ₃,        —OCH₂X¹, —OCHX¹ ₂, —CN, —OH, —SH, —NH₂, -Ph, substituted or        unsubstituted alkyl, substituted or unsubstituted heteroalkyl,        substituted or unsubstituted cycloalkyl, substituted or        unsubstituted heterocycloalkyl, substituted or unsubstituted        aryl, or substituted or unsubstituted heteroaryl;

R^(4B) is hydrogen, —CH₃, —CX² ₃, —CHX² ₂, —CH₂X², —OCX² ₃, —OCH₂X²,—OCHX² ₂, —CN, —OH, —SH, —NH₂, -Ph, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl;

R⁵ is a detectable label or anchor moiety;

R⁶ is hydrogen or a polymerase-compatible cleavable moiety;

R⁷ is hydrogen or —OR^(7A), wherein R^(7A) is hydrogen or apolymerase-compatible cleavable : and X¹ and X² are independentlyhalogen.

Embodiment 2. The compound of embodiment 1, wherein B is a divalentcytosine or a derivative thereof, divalent guanine or a derivativethereof, divalent adenine or a derivative thereof, divalent thymine or aderivative thereof, divalent uracil or a derivative thereof, divalenthypoxanthine or a derivative thereof, divalent xanthine or a derivativethereof, deaza-adenine or a derivative thereof, deaza-guanine or aderivative thereof, deaza-hypoxanthine or a derivative thereof divalent7-methylguanine or a derivative thereof, divalent 5,6-dihydrouracil or aderivative thereof, divalent 5-methylcytosine or a derivative thereof,or divalent 5-hydroxymethylcytosine or a derivative thereof.

Embodiment 3. The compound of embodiment 1, wherein B is

Embodiment 4. The compound of one of embodiments 1 to 3, wherein

L¹ is L^(1A)-L^(1B)-L^(1C)-L^(1D)-L^(1E); and

L^(1A), L^(1B), L^(1C), L^(1D) and L^(1E) are independently a bond,substituted or unsubstituted alkylene, substituted or unsubstitutedheteroalkylene, substituted or unsubstituted cycloalkylene, substitutedor unsubstituted heterocycloalkylene, substituted or unsubstitutedarylene, or substituted or unsubstituted heteroarylene; wherein at leastone of L^(1A), L^(1B), L^(1C), L^(1D) and L^(1E) is not a bond.

Embodiment 5. The compound of one of embodiments 1 to 3, wherein L¹ isL^(1A)-L^(1B)-L^(1C)-L^(1D)-L^(1E); and L^(1A), L^(1B), L^(1C), L^(1D)and L^(1E) are independently a bond, substituted or unsubstituted C₁-C₈alkylene, substituted or unsubstituted 2 to 8 membered heteroalkylene,substituted or unsubstituted C₃-C₈ cycloalkylene, substituted orunsubstituted 3 to 8 membered heterocycloalkylene, substituted orunsubstituted C₆-C₁₀ arylene, or substituted or unsubstituted 5 to 10membered heteroarylene; wherein at least one of L^(1A), L^(1B), L^(1C),L^(1D) and L^(1E) is not a bond.

Embodiment 6. The compound of one of embodiments 1 to 3, wherein L¹ isL^(1A)-L^(1B)-L^(1C)-L^(1D)-L^(1E) and L^(1A), L^(1B), L^(1C), L^(1D)and L^(1E) are independently a bond, substituted or unsubstituted C₁-C₆alkylene, substituted or unsubstituted 2 to 6 membered heteroalkylene,substituted or unsubstituted C₃-C₆ cycloalkylene, substituted orunsubstituted 3 to 6 membered heterocycloalkylene, substituted orunsubstituted phenyl, or substituted or unsubstituted 5 to 6 memberedheteroarylene; wherein at least one of L^(1A), L^(1B), L^(1C), L^(1D)and L^(1E) is not a bond.

Embodiment 7. The compound of one of embodiments 1 to 3, wherein L¹ is abond, substituted or unsubstituted alkylene, substituted orunsubstituted heteroalkylene, substituted or unsubstitutedcycloalkylene, substituted or unsubstituted heterocycloalkylene,substituted or unsubstituted arylene, or substituted or unsubstitutedheteroarylene.

Embodiment 8. The compound of one of claims 1 to 3, wherein L¹ is abond, substituted or unsubstituted C₁-C₈ alkylene, substituted orunsubstituted 2 to 8 membered heteroalkylene, substituted orunsubstituted C₃-C₈ cycloalkylene, substituted or unsubstituted 3 to 8membered heterocycloalkylene, substituted or unsubstituted C₆-C₁₀arylene, or substituted or unsubstituted 5 to 10 membered heteroarylene.

Embodiment 9. The compound of one of embodiments 1 to 3, wherein L¹ is abond, substituted or unsubstituted C₁-C₈ alkylene, substituted orunsubstituted 2 to 8 membered heteroalkylene, substituted orunsubstituted C₃-C₈ cycloalkylene, substituted or unsubstituted 3 to 8membered heterocycloalkylene, substituted or unsubstituted C₆-C₁₀arylene, or substituted or unsubstituted 5 to 10 membered heteroarylene.

Embodiment 10. The compound of one of embodiments 1 to 3, wherein L¹ isa bond, substituted or unsubstituted C₁-C₆ alkylene, substituted orunsubstituted 2 to 6 membered heteroalkylene, substituted orunsubstituted C₃-C₆ cycloalkylene, substituted or unsubstituted 3 to 6membered heterocycloalkylene, substituted or unsubstituted phenyl, orsubstituted or unsubstituted 5 to 6 membered heteroarylene.

Embodiment 11. The compound of one of embodiments 1 to 3, wherein L¹ isa substituted or unsubstituted C₁-C₆ alkylene or substituted orunsubstituted 2 to 6 membered heteroalkylene.

Embodiment 12. The compound of one of embodiments 1 to 3, wherein L¹ isan unsubstituted C₁-C₄ alkylene.

Embodiment 13. The compound of one of 1 to 3, wherein L¹ is —C≡C—CH₂—.

Embodiment 14. The compound of one of embodiments 1 to 12, wherein L² isa cleavable linker.

Embodiment 15. The compound of one of embodiments 1 to 12, wherein L² isa chemically cleavable linker.

Embodiment 16. The compound of one of embodiments 1 to 12, wherein L² isa photocleavable linker, an acid-cleavable linker, a base-cleavablelinker, an oxidant-cleavable linker, a reductant-cleavable linker, or afluoride-cleavable linker.

Embodiment 17. The compound of one of embodiments 1 to 12, wherein L² isa cleavable linker comprising a dialkylketal linker, an azo linker, anallyl linker, a cyanoethyl linker, a1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl linker, or anitrobenzyl linker.

Embodiment 18. The compound of one of embodiments 1 to 12, wherein L² isL^(2A), L^(2B), L^(2C), L^(2D) and L^(2E); and L^(1A), L^(1B), L^(1C),L^(1D) and L^(1E) are independently a bond, —NN—, —NHC(O)—, —C(O)NH—,substituted or unsubstituted alkylene, substituted or unsubstitutedheteroalkylene, substituted or unsubstituted cycloalkylene, substitutedor unsubstituted heterocycloalkylene, substituted or unsubstitutedarylene, or substituted or unsubstituted heteroarylene; wherein at leastone of L^(2A), L^(2B), L^(2C), L^(2D) and L^(2E) is not a bond.

Embodiment 19. The compound of one of embodiments 1 to 12, wherein L² isL^(2A)-L^(2B)-L^(2C)-L^(2D)-L^(2E) and L^(2A), L^(2B), L^(2C), L^(2D)and L^(2E)are independently a bond, —NN—, —NHC(O)—, —C(O)NH—,substituted or unsubstituted C₁-C₂₀ alkylene, substituted orunsubstituted 2 to 20 membered heteroalkylene, substituted orunsubstituted C₃-C2o cycloalkylene, substituted or unsubstituted 3 to 20membered heterocycloalkylene, substituted or unsubstituted C₆-C₂₀arylene, or substituted or unsubstituted 5 to 20 membered heteroarylene;wherein at least one of L^(2A), L^(2B), L^(2C), L^(2D) and L^(2E) is nota bond.

Embodiment 20. The compound of one of embodiments 1 to 12, wherein L² isL^(2A)-L^(2B)-L^(2C)-L^(2D)-L^(2E) and L^(2A), L^(2B), L^(2C), L^(2D)and L^(2E)are independently a bond, —NN—, —NHC(O)—, —C(O)NH—,substituted or unsubstituted C₁-C alkylene, substituted or unsubstituted2 to 10 membered heteroalkylene, substituted or unsubstituted C₃-C₈cycloalkylene, substituted or unsubstituted 3 to 8 memberedheterocycloalkylene, substituted or unsubstituted C₆-C₁₀ arylene, orsubstituted or unsubstituted 5 to 10 membered heteroarylene; wherein atleast one of L^(2A), L^(2B)-L^(2C)-L^(2D)-L^(2E) is not a bond.

Embodiment 21. The compound of one of embodiments 1 to 12, wherein L² isL^(2A)-L^(2B)-L^(2C)-L^(2D)-L^(2E) and L^(2A), L^(2B), L^(2C), L^(2D)and L^(2E) are independently a bond, —NN—, —NHC(O)—, —C(O)NH—,substituted or unsubstituted C₁-C₆ alkylene, substituted orunsubstituted 2 to 6 membered heteroalkylene, substituted orunsubstituted C₃-C₆ cycloalkylene, substituted or unsubstituted 3 to 6membered heterocycloalkylene, substituted or unsubstituted phenyl, orsubstituted or unsubstituted 5 to 6 membered heteroarylene; wherein atleast one of L^(2A), L^(2B), L^(2C), L^(2D) and L^(2E) is not a bond.

Embodiment 22. The compound of one of embodiments 1 to 12, wherein L² isL^(2A)-L^(2B)-L^(2C)-L^(2D)-L^(2E); L^(2A) is a bond, substituted orunsubstituted alkylene, substituted or unsubstituted heteroalkylene;L^(2B) is a bond, substituted or unsubstituted cycloalkylene,substituted or unsubstituted heterocycloalkylene, substituted orunsubstituted arylene, substituted or unsubstituted heteroarylene;L^(2C) is a bond, substituted or unsubstituted cycloalkylene,substituted or unsubstituted heterocycloalkylene, substituted orunsubstituted arylene, substituted or unsubstituted heteroarylene;L^(2D) is a bond, substituted or unsubstituted alkylene, substituted orunsubstituted heteroalkylene; and L^(2E) is a bond, substituted orunsubstituted alkylene, substituted or unsubstituted heteroalkylene,substituted or unsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene; wherein at least one ofL^(2A), L^(2B), L^(2C), L^(2D) and L^(2E) is not a bond.

Embodiment 23. The compound of one of embodiments 1 to 12, wherein L² isa bond, substituted or unsubstituted alkylene, substituted orunsubstituted heteroalkylene, substituted or unsubstitutedcycloalkylene, substituted or unsubstituted heterocycloalkylene,substituted or unsubstituted arylene, or substituted or unsubstitutedheteroarylene.

Embodiment 24. The compound of one of embodiments 1 to 12, wherein L² isa bond, substituted or unsubstituted C₁-C₂₀ alkylene, substituted orunsubstituted 2 to 20 membered heteroalkylene, substituted orunsubstituted C₃-C₂₀ cycloalkylene, substituted or unsubstituted 3 to 20membered heterocycloalkylene, substituted or unsubstituted C₆-C₂₀arylene, or substituted or unsubstituted 5 to 20 membered heteroarylene.

Embodiment 25. The compound of one of embodiments 1 to 12, wherein L² isa bond, substituted or unsubstituted C₁-C₈ alkylene, substituted orunsubstituted 2 to 8 membered heteroalkylene, substituted orunsubstituted C₃-C₈ cycloalkylene, substituted or unsubstituted 3 to 8membered heterocycloalkylene, substituted or unsubstituted C₆-C₁₀arylene, or substituted or unsubstituted 5 to 10 membered heteroarylene.

Embodiment 26. The compound of one of embodiments 1 to 12, wherein L² isa bond, substituted or unsubstituted C₁-C₆ alkylene, substituted orunsubstituted 2 to 6 membered heteroalkylene, substituted orunsubstituted C₃-C₆ cycloalkylene, substituted or unsubstituted 3 to 6membered heterocycloalkylene, substituted or unsubstituted phenyl, orsubstituted or unsubstituted 5 to 6 membered heteroarylene.

Embodiment 27. The compound of one of embodiments 1 to 12, wherein L² isa substituted or unsubstituted 4 to 10 membered heteroalkylene.

Embodiment 28. The compound of one of embodiments 1 to 12, wherein L² isa substituted or unsubstituted 4 to 8 membered heteroalkylene.

Embodiment 29. The compound of one of embodiments 1 to 12, whereinL²—C(CH₃)₂CH₂NHC(O)—.

Embodiment 30. The compound of one of embodiments 1 to 28, wherein R³ is—OH.

Embodiment 31. The compound of one of embodiments 1 to 28, wherein R³ ismonophosphate.

Embodiment 32. The compound of one of embodiments 1 to 28, wherein R³ ispolyphosphate.

Embodiment 33. The compound of one of embodiments 1 to 28, wherein R³ istriphosphate.

Embodiment 34. The compound of one of embodiments 1 to 28, wherein R³ istetraphosphate, pentaphosphate, or hexaphosphate.

Embodiment 35. The compound of one of embodiments 1 to 28, wherein R³ isa residue of a nucleic acid.

Embodiment 36. The compound of one of embodiments 1 to 28, wherein R³ isa 10 to 25 base residue of a nucleic acid.

Embodiment 37. The compound of one of embodiments 1 to 28, wherein R³ isa 10 to 10,000 base residue of a nucleic acid.

Embodiment 38. The compound of one of embodiments 1 to 36, whereinR^(4A) is hydrogen, —CH₃, —CX¹ ₃, —CHX¹ ₂, —CN, -Ph, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or tmsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl.

Embodiment 39. The compound of one of embodiments 1 to 36, whereinR^(4A) is hydrogen, —CH₃, —CX¹ ₃, —CHX¹ ₂, —CN, -Ph, substituted orunsubstituted C₁-C₆ alkyl, substituted or unsubstituted 2 to 6 memberedheteroalkyl, substituted or unsubstituted C₃-C₆ cycloalkyl, substitutedor unsubstituted 3 to 6 membered heterocycloalkyl, substituted orunsubstituted phenyl, or substituted or unsubstituted 5 to 6 memberedheteroaryl.

Embodiment 40. The compound of one of embodiments 1 to 36, whereinR^(4A) is hydrogen.

Embodiment 41. The compound of one of embodiments 1 to 39, whereinR^(4H) is hydrogen, —CH₃, —CX₃, —CHX² ₂, —CH₂X², —CN, -Ph, substitutedor unsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl.

Embodiment 42. The compound of one of embodiments 1 to 39, whereinR^(4B) is hydrogen, —CH₃, —CX² ₃, —CHX² ₂, —CH₂X², —CN, -Ph,H₂,substituted or unsubstituted C₁-C₆ alkyl, substituted or unsubstituted 2to 6 membered heteroalkyl, substituted or unsubstituted C₃-C₆cycloalkyl, substituted or unsubstituted 3 to 6 memberedheterocycloalkyl, substituted or unsubstituted phenyl, or substituted orunsubstituted 5 to 6 membered heteroaryl.

Embodiment 43. The compound of one of embodiments 1 to 39, whereinR^(4B) is hydrogen.

Embodiment 44. The compound of one of embodiments 1 to 42, wherein R⁵ isa detectable label

Embodiment 45. The compound of one of embodiments 1 to 42, wherein R⁵ isa fluorescent dye.

Embodiment 46. The compound of one of embodiments 1 to 42, wherein R⁵ isan anchor moiety.

Embodiment 47. The compound of one of embodiments 1 to 42, wherein R⁵ isa click chemistry reactant moiety.

Embodiment 48. The compound of one of embodiments 1 to 42, wherein R⁵ isa trans-cyclooctene moiety or azide moiety.

Embodiment 49. The compound of one of embodiments 1 to 42, wherein R⁵ isan affinity anchor moiety.

Embodiment 50. The compound of one of embodiments 1 to 42, wherein R⁵ isa biotin moiety.

Embodiment 51. The compound of one of embodiments 1 to 49, wherein R⁶ ishydrogen.

Embodiment 52. The compound of one of embodiments 1 to 49, wherein R⁶ isa polymerase-compatible cleavable moiety.

Embodiment 53. The compound of one of embodiments 1 to 49, wherein R⁶ isa polymerase-compatible cleavable moiety comprising an azido moiety.

Embodiment 54. The compound of one of embodiments 1 to 49, wherein R⁶ isa polymerase-compatible cleavable moiety comprising a dithiol linker.

Embodiment 55. The compound of one of embodiments 1 to 49, wherein R⁶ isa polymerase-compatible cleavable moiety; and the polymerase-compatiblecleavable moiety is —CH₂N₃.

Embodiment 56. The compound of one of embodiments 1 to 49, wherein R⁶ isa polymerase-compatible cleavable moiety; and the polymerase-compatiblecleavable moiety is:

R^(8A) is independently hydrogen, —CH₃, —CX³ ₃, —CHX³ ₂, —CH₂X³, —CN,-Ph, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl;

R^(8B) is independently hydrogen, —CH₃, —CX⁴ ₃, —CHX⁴ ₂, —CH₂X⁴, —CN,-Ph, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl;

R⁹ is independently hydrogen, —CX⁵ ₃, —CHX⁵ ₂, —CH₂X⁵, —OCH₃, —SCH₃,—NHCH₃, —CN, -Ph, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl;

R¹⁰ is independently hydrogen, —CX⁶ ₃, —CHX⁶ ₂, —CH₂X⁶, —OCH₃, —SCH₃,—NHCH₃, —CN, -Ph, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl;

R¹¹ is independently hydrogen, —CX⁷ ₃, —CHX⁷ ₂, —CH₂X⁷, —OCH₃, —SCH₃,—NHCH₃, —CN, -Ph substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl; and

X³, X⁴, X5, X⁶ and X¹ are independently halogen.

Embodiment 57. The compound of one of embodiments 1 to 49, wherein R⁶ isa polymerase-compatible cleavable moiety; and the polymerase-compatiblecleavable moiety is:

R^(8A) is hydrogen, —CH₃, —CX³ ₃, —CHX³ ₂, —CH₂X³, —CN, -Ph, substitutedor unsubstituted C₁-C₆ alkyl, substituted or unsubstituted 2 to 6membered heteroalkyl, substituted or unsubstituted C₃-C₆ cycloalkyl,substituted or unsubstituted 3 to 6 membered heterocycloalkyl,substituted or unsubstituted phenyl, or substituted or unsubstituted 5to 6 membered heteroaryl;

R^(8B) is hydrogen, —CH₃, —CX⁴ ₃, —CHX⁴ ₂, —CH₂X⁴, —CN, -Ph, substitutedor unsubstituted C₁-C₆ alkyl, substituted or unsubstituted 2 to 6membered heteroalkyl, substituted or unsubstituted C₃-C₆ cycloalkyl,substituted or unsubstituted 3 to 6 membered heterocycloalkyl,substituted or unsubstituted phenyl, or substituted or unsubstituted 5to 6 membered heteroaryl;

R⁹ is hydrogen, —CX⁵ ₃, —CHX³ ₂, —CH₂X³, —OCH₃, —SCH₃, —NHCH₃, —CN, -Ph,substituted or unsubstituted C₁-C₆ alkyl, substituted or unsubstituted 2to 6 membered heteroalkyl, substituted or unsubstituted C₃-C₆cycloalkyl, substituted or unsubstituted 3 to 6 memberedheterocycloalkyl, substituted or unsubstituted phenyl, or substituted orunsubstituted 5 to 6 membered heteroaryl;

R¹⁰ is hydrogen, —CX⁶ ₃, —CHX⁶ ₂, —CH₂X⁶, —OCH₃, —SCH₃, —NHCH₃, —CN,-Ph, —OH, —SH, —NH₂, substituted or unsubstituted C₁-C₆ alkyl,substituted or unsubstituted 2 to 6 membered heteroalkyl, substituted orunsubstituted C₃-C₆ cycloalkyl, substituted or unsubstituted 3 to 6membered heterocycloalkyl, substituted or unsubstituted phenyl, orsubstituted or unsubstituted 5 to 6 membered heteroaryl;

R¹¹ is hydrogen, —CX⁷ ₃, —CHX⁷ ₂, —CH₂X⁷, —OCH₃, —SCH₃, —NHCH₃, —CN,-Ph, —OH, —SH, —NH₂, substituted or unsubstituted C₁-C₆ alkyl,substituted or unsubstituted 2 to 6 membered heteroalkyl, substituted orunsubstituted C₃-C₆ cycloalkyl, substituted or unsubstituted 3 to 6membered heterocycloalkyl, substituted or unsubstituted phenyl, orsubstituted or unsubstituted 5 to 6 membered heteroaryl; and

X³, X⁴, X⁵, X⁶ and X⁷ are independently halogen.

Embodiment 58. The compound of one of embodiments 1 to 49, wherein R⁶ isa polymerase-compatible cleavable moiety; and the polymerase-compatiblecleavable moiety is:

R^(8A) and R^(8B) are independently hydrogen or unsubstituted alkyl; andR⁹, R¹⁰, and R¹¹ are independently unsubstituted alkyl or unsubstitutedheteroalkyl.

Embodiment 59. The compound of one of embodiments 1 to 49, wherein R⁶ isa polymerase-compatible cleavable moiety; and the polymerase-compatiblecleavable moiety is:

R^(8A) and R^(8B) are independently hydrogen or unsubstituted C₁-C₄alkyl; and

R⁹, It^(m), and R′ are independently unsubstituted C₁-C₆ alkyl orunsubstituted 2 to 4 membered heteroalkyl.

Embodiment 60. The compound of one of embodiments lto 49, wherein R⁶ isa polymerase-compatible cleavable moiety; and the polymerase-compatiblecleavable moiety is:

R^(8A) and R^(8B) are independently hydrogen; and R⁹, R¹⁰, and R¹¹ areindependently unsubstituted C₁-C₆ alkyl or unsubstituted 2 to 4 memberedheteroalkyl.

Embodiment 61. The compound of one of embodiments 1 to 49, wherein R⁶ isa polymerase-compatible cicavablc moiety; and the polymerase-compatiblecicavablc moiety is:

R^(8A) and R^(8B) are independently hydrogen; and R⁹, R¹⁰, and R¹¹ areindependently unsubstituted methyl or unsubstituted methoxy.

Embodiment 62. The compound of one of embodiments 1 to 49, wherein R⁶ isa polymerase-compatible cleavable moiety; and the polymerase-compatiblecleavable moiety is:

Embodiment 63. The compound of one of embodiments 1 to 49, wherein R⁷ ishydrogen.

Embodiment 64. The compound of one of embodiments 1 to 70, wherein R⁷ is—OR^(7A); and R_(7A) is hydrogen.

Embodiment 65. The compound of one of embodiments 1 to 49, wherein R⁷ is—OR^(7A); and is a polymerase-compatible cleavable moiety.

Embodiment 66. The compound of one of embodiments 1 to 49, wherein R⁷ is—OR^(7A); and R^(7A) is a polymerase-compatible cleavable moietycomprising an azido moiety.

Embodiment 67. The compound of one of embodiments 1 to 49 wherein R⁷ is—OR^(7A); and R^(7A) is a polymerase-compatible cleavable moietycomprising a dithiol linker, an allyl group, or a 2-n itrobenzyl group.

Embodiment 68. The compound of one of embodiments 1 to 49, wherein R⁷ is—OR^(7A); R^(7A) is a polymerase-compatible cleavable moiety; and thepolymerase-compatible cleavable moiety is —CH₂N₃.

Embodiment 69. The compound of one of embodiments 1 to 49, wherein R⁷ is—OR^(7A); R^(7A) is a polymerase-compatible cleavable moiety; and thepolymerase-compatible cleavable moiety is:

R^(8A) is hydrogen, —CH₃, —CX³ ₃, —CHX³ ₂, —CH₂X³, —CN, -Ph, substitutedor unsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl;

R^(8B) is hydrogen, —CH₃, —CX⁴ ₃, —CHX⁴ ₂, —CH₂X⁴, —CN, -Ph, substitutedor unsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl;

R⁹ is hydrogen, —CX⁵ ₃, —CHX⁵ ₂, —CH₂X⁵, —OCH₃, —SCH₃, —NHCH₃, —CN, -Ph,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl;

R¹⁰ is hydrogen, —CX⁶ ₃, —CHX⁶ ₂, —CH₂X⁶, —OCH₃, —SCH₃, —NHCH₃, —CN,-Ph, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl;

R¹¹ is hydrogen, —CX⁷ ₃, —CHX⁷ ₂, —CH₂X⁷, —OCH₃, —SCH₃, —NHCH₃, —CN,-Ph, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; and

X³, X⁴, X⁵, X⁶ and X⁷ are independently halogen.

Embodiment 70. The compound of one of embodiments 1 to 49, wherein R⁷ is—OR^(7A); R^(7A) is a polymerase-compatible cleavable moiety; and thepolymerase-compatible cleavable moiety is:

R^(8A) is hydrogen, —CH₃, —CX³ ₃, —CHX³ ₂, —CH₂X³, —CN, -Ph, substitutedor unsubstituted C₁-C₆ alkyl, substituted or unsubstituted 2 to 6membered heteroalkyl, substituted or unsubstituted C₃-C₆ cycloalkyl,substituted or unsubstituted 3 to 6 membered heterocycloalkyl,substituted or unsubstituted phenyl, or substituted or unsubstituted 5to 6 membered heteroaryl;

R⁸⁵ is hydrogen, —CH₃, —CX⁴ ₃, —CHX⁴ ₂, —CH₂X⁴, —CN, -Ph, substituted orunsubstituted C₁-C₆ alkyl, substituted or unsubstituted 2 to 6 memberedheteroalkyl, substituted or unsubstituted C₃-C₆ cycloalkyl, substitutedor unsubstituted 3 to 6 membered heterocycloalkyl, substituted orunsubstituted phenyl, or substituted or unsubstituted 5 to 6 memberedheteroaryl;

R⁹ is hydrogen, —CX⁵ ₃, —CHX⁵ ₂, —CH₂X⁵, —OCH₃, —SCH₃, —NHCH₃, —CN, -Ph,substituted or unsubstituted C₁-C₆ alkyl, substituted or unsubstituted 2to 6 membered heteroalkyl, substituted or unsubstituted C₃-C₆cycloalkyl, substituted or unsubstituted 3 to 6 memberedheterocycloalkyl, substituted or unsubstituted phenyl, or substituted orunsubstituted 5 to 6 membered heteroaryl;

R¹⁰ is hydrogen, —CX⁶ ₃, —CHX⁶ ₂, —CH₂X⁶, —OCH₃, —SCH₃, —NHCH₃, —CN,-Ph, substituted or unsubstituted C₁-C₆ alkyl, substituted orunsubstituted 2 to 6 membered heteroalkyl, substituted or unsubstitutedC₃-C₆ cycloalkyl, substituted or unsubstituted 3 to 6 memberedheterocycloalkyl, substituted or unsubstituted phenyl, or substituted orunsubstituted 5 to 6 membered heteroaryl;

R¹¹ is hydrogen, —CX⁷ ₃, —CHX⁷ ₂, —CH₂X⁷, —OCH₃, —SCH₃, —NHCH₃, —CN,-Ph, substituted or unsubstituted C₁-C₆ alkyl, substituted orunsubstituted 2 to 6 membered heteroalkyl, substituted or unsubstitutedC₃-C₆ cycloalkyl, substituted or unsubstituted 3 to 6 memberedheterocycloalkyl, substituted or unsubstituted phenyl, or substituted orunsubstituted 5 to 6 membered heteroaryl; and

X³, X⁴, X⁵, X⁶ and X⁷ are independently halogen.

Embodiment 71. The compound of one of embodiments 1 to 70, wherein R⁷ is—OR^(7A); R^(7A) is a polymerase-compatible cleavable moiety; and thepolymerase-compatible cleavable moiety is:

R^(8A), R^(8B), R⁹, R¹⁰, and R¹¹ are independently hydrogen orunsubstituted methyl.

Embodiment 72. The compound of one of embodiments 1 to 70, wherein R⁷ is—OR^(7A); R^(7A) is a polymerase-compatible cleavable; moiety; and thepolymerase-compatible cleavable moiety is:

Embodiment 73. The compound of embodiment 1, having the formula:

wherein m is an integer from 1 to 4.

Embodiment 74. The compound of embodiment 1, having the formula:

Embodiment 75. The compound of one of embodiments 73 to 74, wherein—CR⁹R¹⁰R¹¹ is unsubstituted methyl, unsubstituted ethyl, unsubstitutedpropyl, unsubstituted isopropyl, unsubstituted butyl, or unsubstitutedtent-butyl.

Embodiment 76. The compound of one of embodiments 73 to 74, wherein R⁹is hydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, —OC (CH₃)₃,—OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂,—SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC (CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃,—NHCH₂CH₃, —NHCH₃, or -Ph.

Embodiment 77. The compound of one of embodiments 73 to 74, wherein R¹⁰is hydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, —OC(CH₃)₃,—OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂,—SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃,—NHCH₂CH₃, —NHCH₃, or -Ph.

Embodiment 78. The compound of one of embodiments 73 to 74, wherein R¹¹is hydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, —OC(CH₃)₃,—OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂,—SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃,—NHCH₂CH₃, —NHCH₃, or -Ph.

Embodiment 79. The compound of one of embodiments 73 to 74, whereinR^(8A) is independently hydrogen, deuterium, —C(CH₃)₃, —CH(CH₃)₂,—CH₂CH₂CH₃, —CH₂CH₃, —CH₃, —CX³ ₃, —CHX³ ₂, —CH₂X³, —CN, or -Ph.

Embodiment 80. The compound of one of embodiments 73 to 74, wherein le'independently is independently hydrogen, deuterium, —C(CH₃)₃, —CH(CH₃)₂,—CH₂CH₂CH₃, —CH₂CH₃, —CH₃, —CX⁴ ₃, —CHX⁴ ₂, —CH₂X⁴, —CN, or -Ph.

Embodiment 81. The compound of one of embodiments 73 to 80, wherein—R^(7A) is hydrogen.

Embodiment 82. The compound of one of embodiments 73 to 80, wherein—R^(7A) is

Embodiment 83. The compound of one of embodiments 73 to 80, wherein—R_(7A) is

Embodiment 84. The compound of one of embodiments 73 to 80, wherein—R^(7A) is

Embodiment 85. The compound of one of embodiments 73 to 85 having theformula:

Embodiment 86. The compound of one of embodiments 73 to 85 having theformula:

Embodiment 87. The compound of embodiment 1, having the formula:

wherein m is an integer from 1 to 4.

Embodiment 88. The compound of embodiment 1, having the formula:

Embodiment 89. The compound of one of embodiments 87 to 88, wherein—CR⁹R¹⁰R¹¹ is unsubstituted methyl, unsubstituted ethyl, unsubstitutedpropyl, unsubstituted isopropyl, unsubstituted butyl, or unsubstitutedtert-butyl.

Embodiment 90. The compound of one of embodiments 87 to 88, wherein R⁹is independently hydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃,—CH₃, OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, SC(CH₃)₃,—SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, NHC(CH₃)₃, —NHCH(CH₃)₂,—NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, or -Ph.

Embodiment 91. The compound of one of embodiments 87 to 88, wherein R¹⁰is independently hydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃,—CH₃, OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃,—SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃, —NHCH(CH₃)₂,—NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, or -Ph.

Embodiment 92. The compound of one of embodiments 87 to 88, wherein R¹¹is independently hydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃,—CH₃, —OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃,—SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃; —SCH₃, —NHC(CH₃)₃, —NHCH(CH₃)₂,—NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, or -Ph,

Embodiment 93. The compound of one of embodiments 87 to 88, whereinR^(8A) is independently hydrogen, deuterium, —C(CH₃)₃, —CH(CH₃)₂,—CH₂CH₂CH₃, —CH₂CH₃, —CH₃, —CX³ ₃, —CHX³ ₂, —CH₂X³, —CN, or -Ph.

Embodiment 94. The compound of one of embodiments 87 to 88, whereinwherein R^(8B) is independently hydrogen, denterium, —C(CH₃)₃,—CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, —CX⁴ ₃, —CHX⁴ ₂, —CH₂X⁴, —CN, or-Ph.

Embodiment 95. The compound of one of embodiments 87 to 94 having theformula:

Embodiment 96. The compound of one of embodiments 87 to 95 having theformula:

Embodiment 97. The compound of one of embodiments 77 to 96, wherein-L²-R⁵ is

and z is an integer from 0 to 10.

Embodiment 98. The compound of embodiment 1, having the formula:

wherein m is an integer from 1 to 4.

Embodiment 99. The compound of embodiment 1, having the formula:

Embodiment 100. The compound of one of embodiments 98 to 99, wherein—CR⁹R¹⁰R¹¹ is unsubstituted methyl, unsubstituted ethyl, unsubstitutedpropyl, unsubstituted isopropyl, unsubstituted butyl, or unsubstitutedtert-butyl.

Embodiment 101. The compound of one of embodiments 98 to 99, wherein R⁹is hydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, —OC(CH₃)₃,—OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂.—SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃,—NHCH₂CH₃, —NHCH₃, or -Ph.

Embodiment 102. The compound of one of embodiments 98 to 99, wherein R¹⁰is hydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, —OC(CH₃)₃,—OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂,—SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃,—NHCH₂CH₃, —NHCH₃, or -Ph.

Embodiment 103. The compound of one of embodiments 98 to 99, wherein R¹¹is hydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂C1⁻13, —CH₃,—OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃,—SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃, —NHCH(CH₃)₂,—NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, or -Ph.

Embodiment 104. The compound of one of embodiments 98 to 99, whereinR^(8A) is independently hydrogen, deuterium, —C(CH₃)₃, —CH(CH₃)₂,—CH₂CH₂CH₃, —CH₂CH₃, —CH₃, —CX³ ₃, —CHX³ ₂, —CH₂X³, —CN, or -Ph.

Embodiment 105. The compound of one of embodiments 98 to 99, wherein le'is independently hydrogen, deuterium, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃,—CH₂CH₃, —CH₃, —CX⁴ ₃, —CHX⁴ ₂, —CH₂X⁴, —CN, or -Ph.

Embodiment 106. The compound of one of embodiments 98 to 105, wherein—R^(7A) is hydrogen.

Embodiment 107. The compound of one of embodiments 98 to 105, wherein—R^(7A) is

Embodiment 108. The compound of one of embodiments 98 to 105, wherein—R^(7A) is

Embodiment 109. The compound of one of embodiments 98 to 105, wherein—R^(7A) is

Embodiment 110. The compound of one of embodiments 98 to 105 having theformula:

Embodiment 111. The compound of one of embodiments 115 to 126 having theformula:

Embodiment 112. The compound of embodiment 1, having the formula:

wherein m is an integer from 1 to 4.

Embodiment 113. The compound of embodiment 1, having the formula:

Embodiment 114. The compound of one of embodiments 129 to 130, wherein—CR⁹R¹⁰R¹¹ is unsubstituted methyl, unsubstituted ethyl, unsubstitutedpropyl, unsubstituted isopropyl, unsubstituted butyl, or unsubstitutedten-butyl.

Embodiment 115. The compound of one of embodiments 129 to 130, whereinR⁹ is hydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, —OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂,—SCH₂CH₂CH₃, —SCH₂CH₃, —SCR,, —NHC(CH₃)₃, —NHCH(Cf1₃)₂, —NHCH₂CH₂CH₃,—NHCH₂CH₃, —NHCH₃, or -Ph.

Embodiment 116. The compound of one of embodiments 129 to 130, wherein—R¹⁰ is hydrogen, —C(CH₃)₂, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃,OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃,—SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃, —NHCH(CH₃)₂,—NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, or -Ph.

Embodiment 117. The compound of one of embodiments 129 to 130, wherein—R¹¹ is hydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃,OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃,—SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃. —SCH₃, —NHC(CH₃)₃, —NHCH(CH₃)₂,—NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, or -Ph.

Embodiment 118. The compound of one of embodiments 129 to 130, whereinR^(8A) is hydrogen, deuterium, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃,—CH₃, —CX³ ₃, —CHX³ ₂, —CH₂X³, —CN, or -Ph.

Embodiment 119. The compound of one of embodiments 129 to 130, whereinR^(8B) is hydrogen, deuterium, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃,—CH₃, —CX⁴ ₃, —CHX⁴ ₂, —CH₂X⁴, —CN, or -Ph.

Embodiment 120. The compound of one of embodiments 129 to 136 having theformula:

Embodiment 121. The compound of one of embodiments 115 to 122 having theformula:

Embodiment 122. The compound of one of embodiments 115 to 138, wherein Bis

Embodiment 123. The compound of embodiment 1, having the formula:

wherein m is an integer from 1 to 4.

Embodiment 124. The compound of embodiment 1, having the formula:

Embodiment 125. The compound of one of embodiments 140 to 141, wherein—CR⁹R¹⁰R¹¹ is unsubstituted methyl, unsubstituted ethyl, unsubstitutedpropyl, unsubstituted isopropyl, unsubstituted butyl, or unsubstitutedtert-butyl.

Embodiment 126. The compound of one of embodiments 140 to 141, whereinR⁹ is hydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃,OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃,—SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃, —NHCH(CH₃)₂,—NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, or -Ph.

Embodiment 127. The compound of one of embodiments 140 to 141, whereinR⁹ is hydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃,OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃,—SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃, —NHCH(CH₃)₂,—NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, or -Ph.

Embodiment 128. The compound of one of embodiments 140 to 141, wherein—R¹¹ is hydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃,OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, SC(CH₃)₃,—SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, NHC(CH₃)₃, —NHCH(CH₃)₂,—NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, or -Ph.

Embodiment 129. The compound of one of embodiments 140 to 141, whereinR^(8A) is independently hydrogen, deuterium, —C(CH₃)₃, —CH(CH₃)₂,—CH₂CH₂CH₃, —CH₂CH₃, —CH₃, —CX³ ₃, —CHX³ ₂, —CH₂X³, —CN, or -Ph.

Embodiment 130. The compound of one of embodiments 140 to 141, whereinR^(8B) is independently hydrogen, deuterium, —C(CH₃)₃, —CH(CH₃)₂,—CH₂CH₂CH₃, —CH₂CH₃, —CH_(3 —C X) ⁴ _(3 —CHX) ⁴ ₂ —CH₂X⁴, —CN, or -Ph.

Embodiment 131. The compound of one of embodiments 140 to 147, wherein—R^(7A) is hydrogen.

Embodiment 132. The compound of one of embodiments 140 to 147, wherein—R^(7A) is

Embodiment 133. The compound of one of embodiments 140 to 147, wherein—R^(7A) is

Embodiment 134. The compound of one of embodiments 140 to 147, wherein—R^(7A) is

Embodiment 135. The compound of one of embodiments 140 to 147 having theformula:

Embodiment 136. The compound of one of embodiments 140 to 147 having theformula:

Embodiment 137. The compound of embodiment 1, having the formula:

wherein m is an integer from 1 to 4.

Embodiment 138. The compound of embodiment 1, having the formula:

Embodiment 139. The compound of one of embodiments 154 to 155, wherein—CR⁹R¹⁰R¹¹ is unsubstituted methyl, unsubstituted ethyl, unsubstitutedpropyl, unsubstituted isopropyl, unsubstituted butyl, or unsubstitutedtent-butyl.

Embodiment 140. The compound of one of embodiments 154 to 155, whereinR⁹ is hydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃,OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, SC(CH₃)₃,—SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, NHC(CH₃)₃, —NHCH(CH₃)₂,—NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, or -Ph.

Embodiment 141. The compound of one of embodiments 154 to 155, wherein—R¹⁰ is hydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃,OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃. —OCH₂CH₃, —OCH₃, SC(CH₃)₃,—SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, NHC(CH₃)₃, —NHCH(CH₃)₂,—NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃. or -Ph.

Embodiment 142. The compound of one of embodiments 154 to 155, wherein—R¹¹ is hydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃,OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃,—SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃, —NHCH(CH₃)₂,—NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, or -Ph.

Embodiment 143. The compound of one of embodiments 154 to 155, whereinR^(8A) is hydrogen, deuterium, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃,—CH₃, —CX³ ₃, —CHX³ ₂, —CH₂X³, —CN, or -Ph.

Embodiment 144. The compound of one of embodiments 154 to 155, whereinR^(8B) is hydrogen, deuterium, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃,—CH₃, —CX⁴ ₃, —CHX⁴ ₂, —CH₂X⁴, —CN, or -Ph.

Embodiment 145. The compound of one of embodiments 154 to 161 having theformula:

Embodiment 146. The compound of one of embodiments 154 to 161 having theformula:

Embodiment 147. A composition of the formula:

wherein --- is a non-covalent bond; B is a base;

L¹ is covalent linker;

L² is covalent linker;

L⁴ is a covalent linker;

R³ is —OH, monophosphate, polyphosphate or a nucleic acid;

R^(4A) is hydrogen, —CH₃, —CX¹ ₃, —CHX¹ ₂, —CH₂X¹, —CN, -Ph, substitutedor unsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl;

R^(4B) is hydrogen, —CH₃, —CX² ₃, —CHX² ₂, —CH₂X², —CN, -Ph, substitutedor unsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orun substituted heteroaryl;

R⁵ is an affinity anchor moiety;

R⁶ is hydrogen or a polymerase-compatible cleavable moiety;

R⁷ is hydrogen or —OR^(A), wherein R^(7A) is hydrogen or apolymerase-compatible cleavable moiety;

R¹² is a complementary affinity anchor moiety binder;

R¹³ is a detectable label; and X¹ and X² are independently halogen.

Embodiment 148. The composition of embodiment147, wherein R⁵ is a biotinmoiety and R₁₂ is a streptavidin moiety.

Embodiment 149. The composition of one of embodiments 147 to 148,wherein L⁴ is an orthogonally cleavable linker.

Embodiment 150. The composition of one of embodiments 147 to 148,wherein L⁴ is a cleavable linker.

Embodiment 151. The composition of one of embodiments 147 to 148,wherein L⁴ is a chemically cleavable linker.

Embodiment 152. The composition of one of embodiments 147 to 148,wherein L⁴ is a photocleavable linker, an acid-cleavable linker, abase-cleavable linker, an oxidant-cleavable linker, areductant-cleavable linker, or a fluoride-cleavable linker.

Embodiment 153. The composition of one of embodiments 147 to 148,wherein L⁴ is a cleavable linker comprising a dialkylketal linker, anazo linker, an allyl linker, a cyanoethyl linker, a1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl linker, or anitrobenzyl linker.

Embodiment 154. The composition of one of embodiments 147 to 148,wherein

L⁴ is L^(4A)-L^(4B)-L^(4C)-L^(4D)-L^(2E); and

L^(4A), L^(4B), L^(4C), L^(4D) and L^(4E) are independently a bond,—NN—, —NHC(O)—, —C(O)NH—, substituted or unsubstituted alkylene,substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene; wherein at least one ofL^(4A), L^(4B), L^(4C), L^(4D) and L^(4E) is not a bond.

Embodiment 155. The composition of one of embodiments 147 to 148,wherein L⁴ is L^(4A)-L^(4B)-L^(4C)-L^(4D)-L^(2E); and L^(4A), L^(4B),L^(4C), L^(4D) and L^(4E) are independently a bond, —NN—, —NHC(O)—,—C(O)NH—, substituted or unsubstituted C₁-C₂₀ alkylene, substituted orunsubstituted 2 to 20 membered heteroalkylene, substituted orunsubstituted C₃-C₂₀ cycloalkylene, substituted or unsubstituted 3 to 20membered heterocycloalkylene, substituted or unsubstituted C₆-C₂₀arylene, or substituted or unsubstituted 5 to 20 membered heteroarylene;wherein at least one of L^(4A), L^(4B), L^(4C), L^(4D), and L^(4E) isnot a bond.

Embodiment 156. The composition of one of embodiments 147 to 148,wherein

L⁴ is L^(4A)-L^(4B)-L^(4C)-L^(4D)-L^(2E); and

L^(4A), L^(4B), L^(4C), L^(4D) and L^(4E) are independently a bond,—NN—, —NHC(O)—, —C(O)NH—, substituted or unsubstituted C₁-C₁₀ alkylene,substituted or unsubstituted 2 to 10 membered heteroalkylene,substituted or unsubstituted C₃-C₈ cycloalkylene, substituted orunsubstituted 3 to 8 membered heterocycloalkylene, substituted orunsubstituted C₆-C₁₀ arylene, or substituted or unsubstituted 5 to 10membered heteroarylene; wherein at least one of L^(4A), L^(4B), L^(4C),L^(4D), and L^(4E) is not a bond.

Embodiment 157. The composition of one of embodiments 147 to 148,wherein

L⁴ is L^(4A)-L^(4B)-L^(4C)-L^(4D)-L^(2E); and

L^(4A), L^(4B), L^(4C), L^(4D) and L^(4E) are independently a bond,—NN—, —NHC(O)—, —C(O)NH—, substituted or unsubstituted C₁-C₆ alkylene,substituted or unsubstituted 2 to 6 membered heteroalkylene, substitutedor unsubstituted C₃-C₆ cycloalkylene, substituted or unsubstituted 3 to6 membered heterocycloalkylene, substituted or unsubstituted phenyl, orsubstituted or unsubstituted 5 to 6 membered heteroarylene; wherein atleast one of L^(4A), L^(4B), L^(4C), L^(4D), and L^(4E) is not a bond.

Embodiment 158. The composition of one of embodiments 147 to 148,wherein

L⁴ is L^(4A)-L^(4B)-L^(4C)-L^(4D)-L^(4E);

L^(4A) is a bond, —NN—, —NHC(O)—, —C(O)NH—, substituted or unsubstitutedalkylene, substituted or unsubstituted heteroalkylene;

L^(4B) is a bond, —NN—, —NHC(O)—, —C(O)NH—, substituted or unsubstitutedcycloalkylene, substituted or unsubstituted heterocycloalkylene,substituted or unsubstituted arylene, substituted or unsubstitutedheteroarylene;

L^(4C) is a bond, —NN—, —NHC(O)—, —C(O)NH—, substituted or unsubstitutedcycloalkylene, substituted or unsubstituted heterocycloalkylene,substituted or unsubstituted arylene, substituted or unsubstitutedheteroarylene;

L^(4D) is a bond, —NN—, —NHC(O)—, —C(O)NH—, substituted or unsubstitutedalkylcnc, substituted or unsubstituted heteroalkylene; and

L^(4E) is a bond, —NN—, —NHC(O)—, —C(O)NH—, substituted or unsubstitutedalkylene, substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene; wherein at least one ofL^(4A), L^(4B), L^(4C), L^(4D), and L^(4E) is not a bond.

Embodiment 159. The composition of one of embodiments 147 to 148,wherein L⁴ is a bond, substituted or unsubstituted alkylene, substitutedor unsubstituted heteroalkylene, substituted or unsubstitutedcycloalkylene, substituted or unsubstituted heterocycloalkylene,substituted or un substituted arylene, or substituted or unsubstitutedheteroarylene.

Embodiment 160. The composition of one of embodiments 147 to 148,wherein L⁴ is a bond, substituted or unsubstituted C₁-C₂₀ alkylene,substituted or unsubstituted 2 to 20 membered heteroalkylene,substituted or unsubstituted C₃-C₂₀ cycloalkylene, substituted orunsubstituted 3 to 20 membered heterocycloalkylene, substituted orunsubstituted C₆-C₂₀ arylene, or substituted or unsubstituted 5 to 20membered heteroarylene.

Embodiment 161. The composition of one of embodiments 147 to 148,wherein L⁴ is a bond, substituted or unsubstituted C₁-C₈ alkylene,substituted or unsubstituted 2 to 8 membered heteroalkylene, substitutedor unsubstituted C₃-C₈ cycloalkylene, substituted or unsubstituted 3 to8 membered heterocycloalkylene, substituted or unsubstituted C₆-C₁₀arylene, or substituted or unsubstituted 5 to 10 membered heteroarylene.

Embodiment 162. The composition of one of embodiments 147 to 148,wherein L⁴ is a bond, substituted or unsubstituted C₁-C₆ alkylene,substituted or unsubstituted 2 to 6 membered heteroalkylene, substitutedor unsubstituted C₃-C₆ cycloalkylene, substituted or unsubstituted 3 to6 membered heterocycloalkylene, substituted or unsubstituted phenyl, orsubstituted or unsubstituted 5 to 6 membered heteroarylene.

Embodiment 163. The composition of one of embodiments 147 to 148,wherein L⁴ is a substituted or unsubstituted 3 to 10 memberedheteroalkylene.

Embodiment 164. The composition of one of embodiments 147 to 148,wherein L⁴ is a substituted or unsubstituted 3 to 8 memberedheteroalkylene.

Embodiment 165. The compound of one of embodiments 147 to 164, whereinR¹³ is a fluorescent dye.

Embodiment 166. A compound of the formula:

wherein

-   -   B is a base;    -   L³ is a cleavable linker;    -   R³ is —OH, monophosphate, polyphosphate or a nucleic acid;    -   R⁵ is a detectable label or anchor moiety;    -   R⁷ is hydrogen or —OR^(7A), wherein R^(7A) is hydrogen or

-   -   R^(8A) is hydrogen, —CH₃, —CX³ ₃, —CHX³ ₂, —CH₂X³, —CN, -Ph,        substituted or unsubstituted alkyl, substituted or unsubstituted        heteroalkyl, substituted or unsubstituted cycloalkyl,        substituted or unsubstituted heterocycloalkyl, substituted or        unsubstituted aryl, or substituted or unsubstituted heteroaryl;    -   R^(8B) is hydrogen, —CH₃, —CX⁴ ₃, —CHX⁴ ₂, —CH₂X⁴, —CN,        -Phsubstituted or unsubstituted alkyl, substituted or        unsubstituted heteroalkyl, substituted or unsubstituted        cycloalkyl, substituted or unsubstituted heterocycloalkyl,        substituted or unsubstituted aryl, or substituted or        unsubstituted heteroaryl;

R⁹ is hydrogen, —CX⁵ ₃, —CHX⁵ ₂, —CH₂X⁵, —OCH₃, —SCH₃, —NHCH₃, —CN, -Ph,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl;

R¹⁰ is hydrogen, —CX⁶ ₃, —CHX⁶ ₂, —CH₂X⁶, —OCH₃, —SCH₃, —NHCH₃, —CN,-Ph, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl;

R¹¹ is hydrogen, —CX⁷ ₃, —CHX⁷ ₂, —CH₂X⁷, —OCH₃, —SCH₃, —NHCH₃, —CN,-Ph, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; and X³, X⁴, X⁵, X⁶ and X⁷ areindependently halogen.

Embodiment 167. The compound of embodiment 183, wherein B is a divalentcytosine or a derivative thereof, divalent guanine or a derivativethereof, divalent adenine or a derivative thereof, divalent thyminc or aderivative thereof, divalent uracil or a derivative thereof, divalenthypoxanthine or a derivative thereof, divalent xanthine or a derivativethereof, divalent 7-methylguanine or a derivative thereof, divalent5,6-dihydrouracil or a derivative thereof, divalent 5-methylcytosine ora derivative thereof, or divalent 5-hydroxymethylcytosine or aderivative thereof

Embodiment 168. The compound of embodiment 166, wherein B is

Embodiment 169. The compound of one of embodiments 166 to 168, whereinL³ is

wherein

L¹ is a bond, substituted or unsubstituted a substituted orunsubstituted alkylene, substituted or unsubstituted heteroalkylene,substituted or unsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene;

L² is a bond, substituted or unsubstituted a substituted orunsubstituted alkylene, substituted or unsubstituted heteroalkylene,substituted or unsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, substitutedor unsubstituted heteroarylene, an orthogonally cleavable linker,non-covalent linker or -L^(2A)-L^(2B)-L^(2C)-L^(2D)-, wherein

-   -   L^(2A) is a bond, substituted or unsubstituted a substituted or        unsubstituted alkylene, substituted or unsubstituted        heteroalkylene;    -   L^(2B) is a bond substituted or unsubstituted cycloalkylene,        substituted or unsubstituted heterocycloalkylene, substituted or        unsubstituted arylene, substituted or unsubstituted        heteroarylene;    -   L^(2C) is a bond substituted or unsubstituted cycloalkylene,        substituted or unsubstituted heterocycloalkylene, substituted or        unsubstituted arylene, substituted or unsubstituted        heteroarylene; and    -   L^(2D) is a bond, substituted or unsubstituted a substituted or        unsubstituted alkylene, substituted or unsubstituted        heteroalkylene, wherein at least one of L^(2A), L^(2B), L^(2C),        L^(2D) is not a bond;

R^(4A) is hydrogen, —CH₃, —CX¹ ₃, —CHX¹ ₂, —CH₂X¹, —CN, -Ph, substitutedor unsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl;

R⁴⁻¹¹ is hydrogen, —CH₃, —CX² ₃, —CHX² ₂, —CH₂X², —CN, -Ph, substitutedor unsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; and X¹ and X² are independently halogen.

Embodiment 170. The compound of one of embodiments 166 to 169, whereinL³ is

L¹ is covalent linker;

L² is covalent linker;

R^(4A) is hydrogen, —CH₃, —CX¹ ₃, —CHX¹ ₂, —CH₂X¹, —CN, -Ph, substitutedor unsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl;

R^(4B) is hydrogen, —CH₃, —CX² ₃, —CHX² ₂, —CH₂X², —CN, -Ph, substitutedor unsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl; and

X¹ and X² are independently halogen.

Embodiment 171. The compound of embodiment 170, wherein L¹ isL^(1A)-L^(1B)-L^(1C)-L^(1D)-L^(1E); and L^(1A), L^(1B), L^(1C), L^(1D),and L^(1E) are independently a bond, substituted or unsubstitutedalkylene, substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene; wherein at least one ofL^(1A), L^(1B), L^(1C), L^(1D), and L^(1E) is not a bond.

Embodiment 172. The compound of embodiment 170, wherein isL^(1A)-L^(1B)-L^(1C)-L^(1D)-L^(1E); and L^(1A), L^(1B), L^(1C), L^(1D),and L^(1E) are independently a bond, substituted or unsubstitutedC_(I)-Ca alkylene, substituted or unsubstituted 2 to 8 memberedheteroalkylene, substituted or unsubstituted C₃-C₈ cycloalkylene,substituted or unsubstituted 3 to 8 membered heterocycloalkylene,substituted or unsubstituted C₆-C₁₀ arylene, or substituted orunsubstituted 5 to 10 membered heteroarylene; wherein at least one ofL^(1A), L^(1B), L^(1C), L^(1D), and L^(1E) is not a bond

Embodiment 173. The compound of embodiment 170, wherein L¹ isL^(1A)-L^(1B)-L^(1C)-L^(1D)-L^(1E); and L^(1A), L^(1B), L^(1C), L^(1D),and L^(1E) are independently a bond, substituted or unsubstituted C₁-C₆alkylene, substituted or unsubstituted 2 to 6 membered heteroalkylene,substituted or unsubstituted C₃-C₆ cycloalkylene, substituted orunsubstituted 3 to 6 membered heterocycloalkylene, substituted orunsubstituted phenyl, or substituted or unsubstituted 5 to 6 memberedheteroarylene; wherein at least one of L^(1A), L^(1B), L^(1C), L^(1D),and L^(1E) is not a bond.

Embodiment 174. The compound of embodiment 170, wherein L¹ is a bond,substituted or unsubstituted alkylene, substituted or unsubstitutedheteroalkylene, substituted or unsubstituted cycloalkylene, substitutedor unsubstituted heterocycloalkylene, substituted or unsubstitutedarylene, or substituted or unsubstituted heteroarylene.

Embodiment 175. The compound of embodiment 170, wherein L¹ is a bond,substituted or unsubstituted C₁-C₈ alkylene, substituted orunsubstituted 2 to 8 membered heteroalkylene, substituted orunsubstituted C₃-C₈ cycloalkylene, substituted or unsubstituted 3 to 8membered heterocycloalkylene, substituted or unsubstituted C₆-C₁₀arylene, or substituted or unsubstituted 5 to 10 membered heteroarylene.

Embodiment 176. The compound of embodiment 170, wherein L¹ is a bond,substituted or unsubstituted C₁-C₆ alkylene, substituted orunsubstituted 2 to 6 membered heteroalkylene, substituted orunsubstituted C₃-C₆ cycloalkylene, substituted or unsubstituted 3 to 6membered heterocycloalkylene, substituted or unsubstituted phenyl, orsubstituted or unsubstituted 5 to 6 membered heteroarylene.

Embodiment 177. The compound of embodiment 170, wherein L¹ is asubstituted or unsubstituted C₁-C₆ alkylene or substituted orunsubstituted 2 to 6 membered heteroalkylene.

Embodiment 178. The compound of embodiment 170, wherein L¹ is anunsubstituted C₁-C₄ alkylene.

Embodiment 179. The compound of embodiment 170, wherein L¹ is —C≡C—CH₂—.

Embodiment 180. The compound of one of embodiments 170 to 179, whereinL² is an orthogonally cleavable linker or a non-covalent linker.

Embodiment 181. The compound of one of embodiments 170 to 179, whereinL² is a cleavable linker.

Embodiment 182. The compound of one of embodiments 170 to 179, whereinL² is a chemically cleavable linker.

Embodiment 183. The compound of one of embodiments 170 to 179, whereinL² is a photocleavable linker, an acid-cleavable linker, abase-cleavable linker, an oxidant-cleavable linker, areductant-cleavable linker, or a fluoride-cleavable linker.

Embodiment 184. The compound of one of embodiments 170 to 179, whereinL² is a cleavable linker comprising a dialkylketal linker, an azolinker, an allyl linker, a cyanocthyl linker, a1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl linker, or anitrobenzyl linker.

Embodiment 185. The compound of one of embodiments 170 to 179, wherein

L² is L^(2A)-L^(2B)-L^(2C)-L^(2D)-L^(2E); and

L^(2A), L^(2B), L^(2C), L^(2D), and L^(2E) are independently a —NN—,—NHC(O)—, —C(O)NH—, bond, substituted or unsubstituted alkylene,substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene;

wherein at least one of L2A, L2B_(,) L2C_(,) L^(2D), and L^(2E) is not abond.

Embodiment 186. The compound of one of embodiments 170 to 179, wherein

L² is L^(2A)-L^(2B)-L^(2C)-L^(2D)-L^(2E); and

L^(2A), L^(2B), L^(2C), L^(2D), and L^(2E) are independently a —NN—,—NHC(O)—, —C(O)NH—, substituted or unsubstituted C₁i-C₂₀ alkylene,substituted or unsubstituted 2 to 20 membered heteroalkylene,substituted or unsubstituted C₃-C₂₀ cycloalkylene, substituted orunsubstituted 3 to 20 membered heterocycloalkylene, substituted orunsubstituted C₆-C₂₀ arylene, or substituted or unsubstituted 5 to 20membered heteroarylene; wherein at least one of L^(2A), L^(2B), L^(2C),L^(2D), and L^(2E) is not a bond

Embodiment 187. The compound of one of embodiments 170 to 179, wherein

L² is L^(2A)-L^(2B)-L^(2C)-L^(2D)-L^(2E); and

L^(2A), L^(2B), L^(2C), L^(2D), and L^(2E) are independently a —NN—,—NHC(O)—, —C(O)NH—, substituted or unsubstituted C₁-C₁₀ alkylene,substituted or unsubstituted 2 to 10 membered heteroalkylene,substituted or unsubstituted C₃-C₈ cycloalkylene, substituted orunsubstituted 3 to 8 membered heterocycloalkylene, substituted orunsubstituted C₆-C₁₀ arylene, or substituted or unsubstituted 5 to 10membered heteroarylene;

wherein at least one of L^(2A), L^(2B), L^(2C), L^(2D), and L^(2E) isnot a bond.

Embodiment 188. The compound of one of embodiments 170 to 179, wherein

L² is L^(2A)-L^(2B)-L^(2C)-L^(2D)-L^(2E); and

L^(2A), L^(2B), L^(2C), L^(2D), and L^(2E) are independently a bond,—NN—, —NHC(O)—, —C(O)NH—, substituted or unsubstituted C₁-C₆ alkylene,substituted or unsubstituted 2 to 6 membered heteroalkylene, substitutedor unsubstituted C₃-C₆ cycloalkylene, substituted or unsubstituted 3 to6 membered heterocycloalkylene, substituted or unsubstituted phenyl, orsubstituted or unsubstituted 5 to 6 membered heteroarylene; wherein atleast one of L^(2A), L^(2B), L^(2C), L^(2D), and L^(2E) is not a bond.

Embodiment 189. The compound of one of embodiments 170 to 179, wherein

L² is L^(2A)-L^(2B)-L^(2C)-L^(2D)-L^(2E);

L^(2A) is a bond, substituted or unsubstituted alkylene, substituted orunsubstituted heteroalkylene;

L^(2B) is a bond, substituted or unsubstituted cycloalkylene,substituted or unsubstituted heterocycloalkylene, substituted orunsubstituted arylene, substituted or unsubstituted heteroarylene;

L^(2C) is a bond, substituted or unsubstituted cycloalkylene,substituted or unsubstituted heterocycloalkylene, substituted orunsubstituted arylene, substituted or unsubstituted heteroarylene;

L^(2D) is a bond, substituted or unsubstituted alkylene, substituted orunsubstituted heteroalkylene; and

L^(2E) is a bond, substituted or unsubstituted alkylene, substituted orunsubstituted heteroalkylene, substituted or unsubstitutedcycloalkylene, substituted or unsubstituted heterocycloalkylene,substituted or unsubstituted arylene, or substituted or unsubstitutedheteroarylene; wherein at least one of L^(2A), L^(2B), L^(2C), L^(2D),and L^(2E) is not a bond.

Embodiment 190. The compound of one of embodiments 170 to 179, whereinL² is a bond, substituted or unsubstituted alkylene, substituted orunsubstituted heteroalkylene, substituted or unsubstitutedcycloalkylene, substituted or unsubstituted heterocycloalkylene,substituted or unsubstituted arylene, or substituted or unsubstitutedheteroarylene.

Embodiment 191. The compound of one of embodiments 170 to 179, whereinL² is a bond, substituted or unsubstituted C₁-C₂₀ alkylene, substitutedor unsubstituted 2 to 20 membered heteroalkylene, substituted orunsubstituted C₃-C₂₀ cycloalkylene, substituted or unsubstituted 3 to 20membered heterocycloalkylene, substituted or unsubstituted C₆-C₂₀arylene, or substituted or unsubstituted 5 to 20 membered heteroarylene.

Embodiment 192. The compound of one of embodiments 170 to 179, whereinL² is a bond, substituted or unsubstituted C₁-C₈ alkylene, substitutedor unsubstituted 2 to 8 membered heteroalkylene, substituted orunsubstituted C₃-C₈ cycloalkylene, substituted or unsubstituted 3 to 8membered heterocycloalkylene, substituted or unsubstituted C₆-C₁₀arylene, or substituted or unsubstituted 5 to 10 membered heteroarylene.

Embodiment 193. The compound of one of embodiments 170 to 179, whereinL² is a bond, substituted or unsubstituted C₁-C₆ alkylene, substitutedor unsubstituted 2 to 6 membered heteroalkylene, substituted orunsubstituted C₃-C₆ cycloalkylene, substituted or unsubstituted 3 to 6membered heterocycloalkylene, substituted or unsubstituted phenyl, orsubstituted or unsubstituted 5 to 6 membered heteroarylene.

Embodiment 194. The compound of one of embodiments 170 to 179, whereinL² is a substituted or unsubstituted 4 to 10 membered heteroalkylene.

Embodiment 195. The compound of one of embodiments 170 to 179, whereinL² is a substituted or unsubstituted 4 to 8 membered heteroalkylene.

Embodiment 196. The compound of one of embodiments 170 to 179, whereinL² is —C(CH₃)₂CH₂NHC(O)—.

Embodiment 197. The compound of one of embodiments 166 to 196, whereinR³ is —OH.

Embodiment 198. The compound of one of embodiments 166 to 196, whereinR³ is monophosphate.

Embodiment 199. The compound of one of embodiments 166 to 196, whereinR³ is polyphosphate.

Embodiment 200. The compound of one of embodiments 166 to 196, whereinR³ is triphosphate.

Embodiment 201. The compound of one of embodiments 166 to 196, whereinR³ is tetraphosphate, pentaphosphate, or hexaphosphate.

Embodiment 202. The compound of one of embodiments 166 to 196, whereinR³ is a residue of a nucleic acid.

Embodiment 203. The compound of one of embodiments 166 to 196, whereinR³ is a residue of a 10 to 25 base nucleic acid.

Embodiment 204. The compound of one of embodiments 166 to 196, whereinR³ is a residue of a 10 to 10,000 base nucleic acid.

Embodiment 205. The compound of one of embodiments 170 to 204, whereinR^(4A) is independently hydrogen, —CH₃, —CX¹ ₃, —CHX¹ ₂, —CH₂X¹, —CN,-Ph, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl.

Embodiment 206. The compound of one of embodiments 170 to 204, whereinR^(4A) is independently hydrogen, —CH₃, —CX¹ ₃, —CHX¹ ₂, —CH₂X¹, —CN,-Ph, substituted or unsubstituted C₁-C₆ alkyl, substituted orunsubstituted 2 to 6 membered heteroalkyl, substituted or unsubstitutedC₃-C₆ cycloalkyl, substituted or unsubstituted 3 to 6 memberedheterocycloalkyl, substituted or unsubstituted phenyl, or substituted orunsubstituted 5 to 6 membered heteroaryl.

Embodiment 207. The compound of one of embodiments 170 to 204, whereinR^(4A) is hydrogen.

Embodiment 208. The compound of one of embodiments 170 to 204, whereinR^(4B) is independently hydrogen, —CH₃, —CX² ₃, —CHX² ₂, —CH₂X², —CN,-Ph, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl.

Embodiment 209. The compound of one of embodiments 170 to 204, whereinR^(4B) is independently hydrogen, —CH₃, —CX² ₃, —CHX² ₂, —CH₂X², —CN,-Ph, substituted or unsubstituted C₁-C₆ alkyl, substituted orunsubstituted 2 to 6 membered heteroalkyl, substituted or unsubstitutedC₃-C₆ cycloalkyl, substituted or unsubstituted 3 to 6 memberedheterocycloalkyl, substituted or unsubstituted phenyl, or substituted orunsubstituted 5 to 6 membered heteroaryl.

Embodiment 210. The compound of one of embodiments 170 to 204, whereinR^(4B) is hydrogen.

Embodiment 211. The compound of one of embodiments 166 to 210, whereinR⁵ is a detectable label

Embodiment 212. The compound of one of embodiments 166 to 210, whereinR⁵ is a fluorescent dye.

Embodiment 213. The compound of one of embodiments 166 to 210, whereinR⁵ is an anchor moiety.

Embodiment 214. The compound of one of embodiments 166 to 210, whereinR⁵ is a click chemistry reactant moiety.

Embodiment 215. The compound of one of embodiments 166 to 210, whereinR⁵ is a trans-cyclooctene moiety or azide moiety.

Embodiment 216. The compound of one of embodiments 166 to 210, whereinR⁵ is an affinity anchor moiety.

Embodiment 217. The compound of one of embodiments 166 to 210, whereinR⁵ is a biotin moiety.

Embodiment 218. The compound of one of embodiments 166 to 217, wherein

R^(8A) is hydrogen, —CH₃, —CX³ ₃, —CHX³ ₂, —CH₂X³, —CN, -Ph, substitutedor unsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl;

R^(8B) is hydrogen, —CH₃, —CX⁴ ₃, —CHX⁴ ₂, —CH₂X⁴, —CN, -Ph, substitutedor unsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl;

R⁹ is hydrogen, —CX⁵ ₃, —CHX⁵ ₂, —CH₂X⁵, —OCH₃, —SCH₃, —NHCH₃, —CN, -Ph,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl;

R¹⁰ is hydrogen, —CX⁶ ₃, —CHX⁶ ₂, —CH₂X⁶, —OCH₃, —SCH₃, —NHCH₃, —CN,-Ph, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl;

R¹¹ is hydrogen, —CX⁷ ₃, —CHX⁷ ₂, —CH₂X⁷, —OCH₃, —SCH₃, —NHCH₃, —CN,-Ph, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; and

X³, X⁴, X⁵, X⁶ and X⁷ are independently halogen.

Embodiment 219. The compound of one of embodiments 166 to 217, wherein

R^(8A) and R^(8B) are independently hydrogen or unsubstituted alkyl; and

R⁹, R¹⁰,and R¹¹ are independently unsubstituted alkyl or unsubstitutedheteroalkyl.

Embodiment 220. The compound of one of embodiments 166 to 217, wherein

R^(8A) and R^(8B) are independently hydrogen or unsubstituted C₁-C₄alkyl; and

R⁹, R¹⁰, and R¹¹ are independently unsubstituted C₁-C₆ alkyl orunsubstituted 2 to 4 membered heteroalkyl.

Embodiment 221. The compound of one of embodiments 166 to 217, wherein

R^(8A) and R^(8B) are independently hydrogen; and

R⁹, R¹⁰,and R¹¹ are independently unsubstituted C₁-C₆ alkyl orunsubstituted 2 to 4 membered heteroalkyl.

Embodiment 222. The compound of one of embodiments 166 to 217, wherein

R^(8A) and R^(8B) are independently hydrogen; and

R⁹, R¹⁰ , and R¹¹ are independently unsubstituted methyl orunsubstituted methoxy.

Embodiment 223. The compound of one of embodiments 166 to 217, whereinR^(8A), R^(8B),

R⁹, R¹⁰, and R¹¹ are independently hydrogen or unsubstituted methyl.

Embodiment 224. The compound of one of embodiments 166 to 217, whereinR^(8A) and R^(8B) are hydrogen and R⁹, R¹⁰, R¹¹ are unsubstitutedmethyl.

Embodiment 225. The compound of one of embodiments 166 to 217, whereinR⁷ is hydrogen.

Embodiment 226. The compound of one of embodiments 166 to 217, wherein127 is —OR^(7A); and R^(7A) is hydrogen.

Embodiment 227. The compound of one of embodiments 166 to 217, whereinR⁷ is —OR^(7A) and R^(7A) is:

Embodiment 228. The compound of one of embodiments 166 to 227, wherein127 is —OR^(7A) and R^(7A) is:

Embodiment 229. The compound of embodiment 166, having the formula:

wherein m is an integer from 1 to 4.

Embodiment 230. The compound of embodiment 166, having the formula:

Embodiment 231. The compound of one of embodiments 229 to 230, wherein—CR⁹R¹° R¹¹ is unsubstituted methyl, unsubstituted ethyl, unsubstitutedpropyl, unsubstituted isopropyl, unsubstituted butyl, or unsubstitutedtert-butyl.

Embodiment 232. The compound of one of embodiments 229 to 230, whereinR⁹ is hydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃,OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃,—SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC (CH₃)₃, —NHCH(CH₃)₂,—NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃. or -Ph.

Embodiment 233. The compound of one of embodiments 229 to 230, whereinR¹⁰ is hydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃,OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃,—SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃, —NHCH(CH₃)₂,—NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃. or -Ph.

Embodiment 234. The compound of one of embodiments 229 to 230, whereinR¹¹ is hydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃,OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃,—SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃, —NHCH(CH₃)₂,—NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, or -Ph.

Embodiment 235. The compound of one of embodiments 229 to 230, whereinR^(8A) is hydrogen, deuterium, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃,—CH₃, —CX³ ₃, —CHX³ ₂, —CH₂X³, —CN, -Ph.

Embodiment 236. The compound of one of embodiments 229 to 230, whereinR^(8B) is hydrogen, deuterium, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃,—CH₃, —CX⁴ ₃, —CHX⁴ ₂, —CH₂X⁴, —CN, -Ph.

Embodiment 237. The compound of one of embodiments 229 to 236, wherein—R^(7A) is hydrogen.

Embodiment 238. The compound of one of embodiments 229 to 236, wherein—R^(7A) is

Embodiment 239. The compound of one of embodiments 229 to 236, wherein—R^(&A) is

Embodiment 240. The compound of one of embodiments 229 to 236, wherein—R^(7A) is

Embodiment 241. The compound of one of embodiments 229 to 236 having theformula:

Embodiment 242. The compound of one of embodiments 229 to 236 having theformula:

Embodiment 243. The compound of one of embodiments 229 to 242, wherein Bis

Embodiment 244. The compound of one of embodiments 229 to 243, whereinR⁵ is

Embodiment 245. The compound of embodiment 166. having the formula:

wherein m is an integer from 1 to 4.

Embodiment 246. The compound of embodiment 166, having the formula:

Embodiment 247. The compound of one of embodiments 245 to 246, wherein—CR⁹R¹⁰R¹¹ is unsubstituted methyl, unsubstituted ethyl, unsubstitutedpropyl, unsubstituted isopropyl, unsubstituted butyl, or unsubstitutedtent-butyl.

Embodiment 248. The compound of one of embodiments 245 to 246, whereinR⁹ is hydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃,OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃,—SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃, —NHCH(CH₃)₂,—NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, or -Ph.

Embodiment 249. The compound of one of embodiments 245 to 246, whereinR¹⁰ is hydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃,OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃,—SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃. —SCH₃, —NHC(CH₃)₃, —NHCH(CH₃)₂,—NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, or -Ph.

Embodiment 250. The compound of one of embodiments 245 to 246, whereinR¹¹ is hydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃,OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃,—SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC (CH₃)₃, —NHCH(CH₃)₂,—NHCH₂CH₂CH₃, —NHCH₂ CH₃, —NHCH₃, or -Ph.

Embodiment 251. The compound of one of embodiments 245 to 246, whereinR^(8A) is hydrogen, deuterium, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃,—CH₃, —CX³ ₃, —CHX³ ₂, —CH₂X³, —CN, -Ph.

Embodiment 252. The compound of one of embodiments 245 to 246, whereinR^(8B) is hydrogen, deuterium, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃,—CH₃, —CX⁴ ₃, —CHX⁴ ₂, —CH₂X⁴, —CN, -Ph.

Embodiment 253. The compound of one of embodiments 245 to 246 having theformula:

Embodiment 254. The compound of one of embodiments 242 to 246 having theformula:

Embodiment 255. The compound of one of embodiments 229 to 254, wherein Bis

Embodiment 256, The compound of one of embodiments 229 to 255, whereinR^(s) is

Embodiment 257. A compound of the formula:

wherein

R^(7A) is hydrogen or a polymerase-compatible cleavable moiety;

R^(8A) is hydrogen, —CX³ ₃, —CHX³ ₂, —CH₂X³, —OCX³ ₃, —OCH₂X³, —OCHX³ ₂,—CN, —OH, —SH, —NH₂, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl;

R^(8B) is hydrogen, —CX⁴ ₃, —CHX⁴ ₂, —CH₂X⁴. —OCX⁴ ₃, —OCH₂X⁴, —OCHX⁴ ₂,—CN, —OH, —SH, —NH₂, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl;

R⁹ is hydrogen, —CX⁵ ₃, —CHX⁵ ₂, —CH₂X⁵, —OCX⁵ ₃, —OCH₂X⁵, —OCHX⁵ ₂,—CN, —OH, —SH, —NH₂, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl;

R^(th) is hydrogen, —CX⁶ ₃, —CHX⁶ ₂, —CH₂X⁶, —OCX⁶ ₃, —OCH₂X⁶, —OCHX⁶ ₂,—CN, —OH, —SH, —NH₂, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl;

R¹¹ is hydrogen, —CX⁷ ₃, —CHX⁷ ₂, —CH₂X⁷, —OCX⁷ ₃, —OCH₂X⁷, —OCHX⁷ ₂,—CN, —OH, —SH, —NH₂, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl; and

X³, X⁴, X⁵, X⁶ and X⁷ are independently halogen; and m is an integerfrom 1 to 4.

Embodiment 258. A compound of the formula:

wherein

R7^(A) is hydrogen or a polymerase-compatible cleavable moiety; R^(8A)is independently hydrogen, —CX³ ₃, —CHX³ ₂, —CH₂X³, —OCH₂X³, —OCHX³ ₂,—CN, —OH, —SH, —NH₂, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted aryl, or substituted or unsubstituted heteroaryl;

R^(8B) is independently hydrogen, —CX⁴ ₃, —CHX⁴ ₂, —CH₂X⁴, —OCX⁴ ₃,—OCH₂X⁴, —OCHX⁴ ₂, —CN, —OH, —SH, —NH₂, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl;

R⁹ is independently hydrogen, —CX⁵ ₃, —CHX⁵ ₂, —CH₂X⁵, —OCX⁵ ₃, —OCH₂X⁵,—OCHX⁵ ₂, —CN, —OH, —SH, —NH₂, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl;

R¹⁰ is independently hydrogen, —CX⁶ ₃, —CHX⁶ ₂, —CH₂X⁶, —OCX⁶ ₃,—OCH₂X⁶, —OCHX⁶ ₂, —CN, —OH, —SH, —NH₂, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl;

R¹¹ is independently hydrogen, —CX⁷ ₃, —CHX⁷ ₂, —CH₂X⁷, —OCX⁷ ₃,—OCH₂X⁷, —OCHX⁷ ₂, —CN, —OH, —SH, —NH₂, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl; and

X³, X⁴, X⁵, X⁶ and X⁷ are independently halogen.

Embodiment 259. The compound of one of embodiments 257 to 258, wherein—CR⁹R¹⁰R¹¹ is unsubstituted methyl, unsubstituted ethyl, unsubstitutedpropyl, unsubstituted isopropyl, unsubstituted butyl, or unsubstitutedtent-butyl.

Embodiment 260. The compound of one of embodiments 257 to 258, whereinR⁹ is hydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃,OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃,—SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃, —NHCH(CH₃)₂,—NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, or -Ph.

Embodiment 261. The compound of one of embodiments 257 to 258, whereinR¹⁰ is hydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃, —OCH(CH₃)₂, CH₂CH₂ CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂,SCH₂ CH₂CH₃ SCH₂CH₃, —SCH₃, —NHC(CH₃)₃, —NHCH(CH₃)₂, —NHCH₂CH₂CH₃,—NHCH₂CH₃, —NHCH₃, or -Ph.

Embodiment 262. The compound of one of embodiments 257 to 258, whereinR¹¹ is hydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃,OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃,—SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃, —NHCH(CH₃)₂,—NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, or -Ph.

Embodiment 263. The compound of one of embodiments 257 to 258, whereinR^(8A) is hydrogen, deuterium, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃,—CH₃, —CX³ ₃, —CHX³ ₂, —CH₂X³, —CN, -Ph.

Embodiment 264. The compound of one of embodiments 257 to 258, whereinR^(8B) is hydrogen, deuterium, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃,-CH₃, —CX⁴ ₃, —CHX⁴ ₂, —CH₂X⁴, —CN, -Ph.

Embodiment 265. The compound of one of embodiments 257 to 258, wherein—R^(7A) is hydrogen.

Embodiment 266. The compound of one of embodiments 257 to 258, wherein—R^(7A) is

Embodiment 267. The compound of one of embodiments 257 to 258, wherein—R^(7A) is

Embodiment 268. The compound of one of embodiments 257 to 258, wherein—R^(7A) is

Embodiment 269. The compound of one of embodiments 257 to 258 having theformula:

Embodiment 270. A compound of the formula:

wherein

R^(8A) is independently hydrogen, CH₃, —CX³ ₃, —CHX³ ₂, —CH₂X³, —OCX³ ₃,—OCH₂X³, —OCHX³ ₂, —CN, -Ph —OH, —SH, —NH₂, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl;

R^(8B) is independently hydrogen, CH₃, —CX⁴ ₃, —CHX⁴ ₂, —CH₂X⁴, —OCX⁴ ₃,—OCH₂X⁴, —OCHX⁴ ₂, —CN, -Ph —OH, —SH, —NH₂, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl;

R⁹ is independently hydrogen, —CX⁵ ₃, —CHX⁵ ₂, —CH₂X⁵, —OCX⁵ ₃, —OCH₂X⁵,—OCHX⁵ ₂, —CN, —OH, —SH, —NH₂, substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heterocycloalkyl, substitutedor unsubstituted aryl, or substituted or unsubstituted heteroaryl;

R¹⁰ is independently hydrogen, —CX⁶ ₃, —CHX⁶ ₂, —CH₂X⁶, —OCX⁶ ₃,—OCH₂X⁶, —OCHX⁶ ₂, —CN, —OH, —SH, —NH₂, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl;

R¹¹ is independently hydrogen, —CX⁷ ₃, —CHX⁷ ₂, —CH₂X⁷, —OCX⁷ ₃,—OCH₂X⁷, —OCHX⁷ ₂, —CN, —OH, —SH, —NH₂, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroary; and

X³, X⁴, X⁵, X⁶ and X⁷ are independently halogen; and m is an integerfrom 1 to 4.

Embodiment 271. A compound of the formula:

wherein

R^(8A) is independently hydrogen, CH₃, —CX³ ₃, —CHX³ ₂, —CH₂X³, —OCX³ ₃,—OCH₂X³, —OCHX³ ₂, —CN, -Ph —OH, —SH, —NH₂, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl;

R^(8B) is independently hydrogen, CH₃, —CX⁴ ₃, —CHX⁴ ₂, —CH₂X⁴, —OCX⁴ ₃,—OCH₂X⁴, —OCHX⁴ ₂, —CN, -Ph, —OH, —SH, —NH₂, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted aryl, or substituted orunsubstituted heteroaryl;

R⁹ is independently hydrogen, —CX⁵ ₃, —CHX⁵ ₂, —CH₂X⁵, -0 CX⁵ ₃,—OCH₂X⁵, —OCHX⁵ ₂, —CN, —OH, —SH, —NH₂, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl;

R¹⁰ is independently hydrogen, —CX⁶ ₃, —CHX⁶ ₂, —CH₂X⁶, —OCX⁶ ₃,—OCH₂X⁶, —OCHX⁶ ₂, —CN, —OH, —SH, —NH₂, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl;

R¹¹ is independently hydrogen, —CX⁷ ₃, —CHX⁷ ₂, —CH₂X⁷, —OCX⁷ ₃,—OCH₂X⁷, —OCHX⁷ ₂, —CN, —OH, —SH, —NH₂, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl; and X³, X¹, X⁵, X⁶ and X⁷ are independently halogen.

Embodiment 272. The compound of one of embodiments 270 to 271, wherein—CR⁹R¹⁰R¹¹ is unsubstituted methyl, unsubstituted ethyl, unsubstitutedpropyl, unsubstituted isopropyl, unsubstituted butyl, or unsubstitutedtent-butyl.

Embodiment 273. The compound of one of embodiments 270 to 271, whereinR⁹ is hydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃,OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃,—SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃, —NHCH(CH₃)₂,—NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, or -Ph.

Embodiment 274. The compound of one of embodiments 270 to 271, wherein—R¹⁰ is hydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃,OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, -OCH₂CH₃, —OCH₃, —SC(CH)₃,—SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃, —NHCH(CH₃)₂,—NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, or -Ph.

Embodiment 275. The compound of one of embodiments 270 to 271, wherein—R″ is hydrogen, —C(CH₃)₃, —CH(CH₃)₂, —CH₂CH₂CH₃, —CH₂CH₃, —CH₃,OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃, —OCH₃, —SC(CH₃)₃,—SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃, —NHCH(CH₃)₂,—NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, or -Ph.

Embodiment 276. The compound of one of embodiments 270 to 271, whereinR^(8A) is independently hydrogen, deuterium, —C(CH₃)₃, —CH(CH₃)₂,—CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃,—OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃,—NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, or -Ph.

Embodiment 277. The compound of one of embodiments 270 to 271, whereinR^(8B) is independently hydrogen, deuterium, —C(CH₃)₃, —CH(CH₃)₂,—CH₂CH₂CH₃, —CH₂CH₃, —CH₃, OC(CH₃)₃, —OCH(CH₃)₂, —OCH₂CH₂CH₃, —OCH₂CH₃,—OCH₃, —SC(CH₃)₃, —SCH(CH₃)₂, —SCH₂CH₂CH₃, —SCH₂CH₃, —SCH₃, —NHC(CH₃)₃,—NHCH(CH₃)₂, —NHCH₂CH₂CH₃, —NHCH₂CH₃, —NHCH₃, or -Ph.

Embodiment 278. The compound of one of embodiments 271 to 277 having theformula:

Embodiment 279. A compound of the formula:

R^(12z)-L^(4z)-R¹³;

wherein

L^(4z) is a covalent linker;

R^(12z) is a complementary anchor moiety reactive group; and

R¹³ is a detectable label.

Embodiment 280. The compound of embodiment 279, wherein L^(4z) is anorthogonally cleavable linker.

Embodiment 281. The compound of embodiment 279, wherein L^(4z) is acleavable linker.

Embodiment 282. The compound of embodiment 279, wherein L^(4z) is achemically cleavable linker.

Embodiment 283. The compound of embodiment 279, wherein L^(4z) is aphotocleavable linker, an acid-cleavable linker, a base-cleavablelinker, an oxidant-cleavable linker, a reductant-cleavable linker, or afluoride-cleavable linker.

Embodiment 284. The compound of embodiment 279, wherein L^(4z) is acleavable linker comprising a dialkylketal linker, an azo linker, anallyl linker, a cyanoethyl linker, a1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl linker, or anitrobenzyl linker.

Embodiment 285. The compound of embodiment 279, wherein

L^(4zA)-L^(4zB)-L^(4zC)-L^(4zD)-L^(4zE); and

L^(4zA), L^(4zB), L^(4zC), L^(4zD), and L^(4zE) are independently abond, —NN—, —NHC(O)—, —C(O)NH—, substituted or unsubstituted alkylene,substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene;

wherein at least one of L^(4z) ^(A), L4zB, ozc_(,) ozp, _(an)d L^(4z)^(E) i_(s no)t _(a) b_(o)nd.

Embodiment 286. The compound of embodiment 279, wherein

L^(4z) is L4zA-L4zB-ozc_ozD_ozc_(; and) ozA_(, L)4z13_(, L)4zC_(,)1,^(4zD), and L^(4z) ^(E) are independently a bond, —NN—, —NHC(O)—,—C(O)NH—, substituted or unsubstituted C₁-C₂₀ alkylene, substituted orunsubstituted 2 to 20 membered heteroalkylene, substituted orunsubstituted C₃-C₂₀ cycloalkylene, substituted or unsubstituted 3 to 20membered heterocycloalkylene, substituted or unsubstituted C₆-C₂₀arylene, or substituted or unsubstituted 5 to 20 membered heteroarylene;

wherein at least one of L^(4zA), L^(4zB), L^(4zC), L^(4zD), and L^(4zE)is not a bond.

Embodiment 287. The compound of embodiment 279, wherein

L^(4zA)-L^(4zB)-L^(4zC)-L^(4zD)-L^(4zE); and

L^(4zA), L^(4zB), L^(4zC), L^(4zD), and L^(4zE) are independently abond, —NN—, —NHC(O)—, —C(O)NH—, substituted or unsubstituted C₁-C₁₀alkylene, substituted or unsubstituted 2 to 10 membered heteroalkylene,substituted or unsubstituted C₃-C₈ cycloalkylene, substituted orunsubstituted 3 to 8 membered heterocycloalkylene, substituted orunsubstituted C₆-C₁₀ arylene, or substituted or unsubstituted 5 to 10membered heteroarvlene;

wherein at least one of L^(4zA), L^(4zB), L^(4zC), L^(4zD), and L^(4zE)is not a bond.

Embodiment 288. The compound of embodiment 279, wherein

L^(4zA)-L^(4zB)-L^(4zC)-L^(4zD)-L^(4zE); and

L^(4zA), L^(4zB), L^(4zC), L^(4zD), and L^(4zE) are independently abond, —NN—, —NHC(O)—, —C(O)NH—, substituted or unsubstituted C₁-C₆alkylene, substituted or unsubstituted 2 to 6 membered heteroalkylene,substituted or unsubstituted C₃-C₆ cycloalkylene, substituted orunsubstituted 3 to 6 membered heterocycloalkylene, substituted orunsubstituted phenyl, or substituted or unsubstituted 5 to 6 memberedheteroarylene;

wherein at least one of L^(4zA), L^(4zB), L^(4zC), L^(4zD), and L^(4zE)is not a bond.

Embodiment 289. The compound of embodiment 279, wherein

L^(4z) is L^(4zA)-L^(4zB)-L^(4zC)-L^(4zD)-L^(4zE);

L^(4zA) is a bond, —NN—, —NHC(O)—, —C(O)NH—, substituted orunsubstituted alkylene, substituted or unsubstituted heteroalkylene;

L^(4zB) is a bond, —NN—, —NHC(O)—, —C(O)NH—, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedhacrocycloalkylenc, substituted or unsubstituted arylcnc, substituted orunsubstituted heteroarylene;

L^(4zC) is a bond, —NN—, —NHC(O)—, —C(O)NH—, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, substitutedor unsubstituted heteroarylene;

L^(4zD) is a bond, —NN—, —NHC(O)—, —C(O)NH—, substituted orunsubstituted alkylene, substituted or unsubstituted heteroalkylene; and

L^(4zE) is a bond, —NN—, —NHC(O)—, —C(O)NH—, substituted orunsubstituted alkylene, substituted or unsubstituted heteroalkylene,substituted or unsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene;

wherein at least one of L^(4zA), L^(4zB), L^(4zC), L^(4zD), and L^(4zE)is not a bond.

Embodiment 290. The compound of embodiment 279, wherein L^(4z) is abond, substituted or unsubstituted alkylene, substituted orunsubstituted heteroalkylene, substituted or unsubstitutedcycloalkylene, substituted or unsubstituted heterocycloalkylene,substituted or unsubstituted arylene, or substituted or unsubstitutedheteroarylene.

Embodiment 291. The compound of embodiment 279, wherein L^(4z) is abond, substituted or unsubstituted C₁-C₂₀ alkylene, substituted orunsubstituted 2 to 20 membered heteroalkylene, substituted orunsubstituted C₃-C₂₀ cycloalkylene, substituted or unsubstituted 3 to 20membered heterocycloalkylene, substituted or unsubstituted C₆-C₂₀arylene, or substituted or unsubstituted 5 to 20 membered heteroarylene.

Embodiment 292. The compound of embodiment 279, wherein L^(4z) is abond, substituted or unsubstituted C₁-C₈ alkylene, substituted orunsubstituted 2 to 8 membered heteroalkylene, substituted orunsubstituted C₃-C₈ cycloalkylene, substituted or unsubstituted 3 to 8membered heterocycloalkylene, substituted or unsubstituted C₆-C₁₀arylene, or substituted or unsubstituted 5 to 10 membered heteroarylene.

Embodiment 293. The compound of embodiment 279, wherein L^(4z) is abond, substituted or unsubstituted C₁-C₆ alkylene, substituted orunsubstituted 2 to 6 membered heteroalkylene, substituted orunsubstituted C₃-C₆ cycloalkylene, substituted or unsubstituted 3 to 6membered heterocycloalkylene, substituted or unsubstituted phenyl, orsubstituted or unsubstituted 5 to 6 membered heteroarylene.

Embodiment 294. The compound of embodiment 279, wherein L^(4z) is asubstituted or unsubstituted 3 to 10 membered heteroalkylene.

Embodiment 295. The compound of embodiment 279, wherein L^(4z) is asubstituted or unsubstituted 3 to 8 membered heteroalkylene.

Embodiment 296. The compound of embodiment 279 having the formula:

wherein

z is an integer from 0 to 20.

Embodiment 297. The compound of one of embodiments 279 to 296, whereinR^(12z) is

a streptavidin moiety, or

Embodiment 298. The compound of one of embodiments 279 to 296, whereinR¹¹ is a fluorescent dye.

Embodiment 299. The compound of one of embodiments 279 to 296, whereinR¹³ comprises a fluorescence resonance energy transfer donor fluorescentdye.

Embodiment 300. The compound of one of embodiments 279 to 296, whereinR^(—)comprises a fluorescence resonance energy transfer acceptorfluorescent dye.

Embodiment 301. The compound of one of embodiments 279 to 296, whereinR¹³ comprises a fluorescence resonance energy transfer donor andacceptor fluorescent dye pair connected by a linker.

Embodiment 302. The compound of one of embodiments 279 to 296, whereinR¹³ comprises a fluorescence resonance energy transfer donor andacceptor fluorescent dye pair connected by a linker and separated byfrom 0.1 nm to 10 nm.

Embodiment 303. The compound of one of embodiments 279 to 296, whereinR″ is

Embodiment 304. The compound of embodiment 296 having the formula:

Embodiment 305. A compound of the formula:

R^(12z)—R¹⁴;

wherein

R^(12z) is a complementary anchor moiety reactive group; and

R¹⁴ -substituted alkyl, R¹⁵-substituted heteroalkyl, R¹⁵-substitutedcycloalkyl, R¹⁵-substituted heterocycloalkyl, R¹⁵-substituted aryl, orR¹⁵-substituted heteroaryl;

R¹⁵ is independently R¹⁶-substituted alkyl, R¹⁶-substituted heteroalkyl,R¹⁶-substituted cycloalkyl, R¹⁶-substituted heterocycloalkyl,R¹⁶-substituted aryl, R¹⁶-substituted heteroaryl, or a detectable dye;

R¹⁶ is independently R¹⁷-substituted alkyl, R¹⁷-substituted heteroalkyl,R¹⁷-substituted cycloalkyl, R¹⁷-substituted heterocycloalkyl,R¹⁷-substituted aryl, R¹⁷-substituted heteroaryl, or a detectable dye;

R¹⁷ is independently R¹⁸-substituted alkyl, R¹⁸-substituted heteroalkyl,R¹⁸-substituted cycloalkyl, R¹⁸-substitutcd hctcrocycloalkyl,R¹⁸-substitutcd aryl, R¹⁸-substitutcd hctcroaryl, or a detectable dye;

R¹⁸ is a detectable dye;

wherein R¹⁴ is substituted with a plurality of R¹⁵ moieties, R¹⁵ issubstituted with a plurality of R¹⁶ moieties, and le is substituted witha plurality of R¹⁷ moieties.

Embodiment 305. The compound of embodiment 304, wherein R^(12z) is

a streptavidin moiety, or

Embodiment 306. The compound of one of embodiments 304 to 305, whereinthe detectable dye is a fluorescent dye.

Embodiment 307. The compound of one of embodiments 304 to 305, whereinthe detectable dye comprises a fluorescence resonance energy transferdonor fluorescent dye.

Embodiment 308. The compound of one of embodiments 304 to 305, whereinthe detectable dye comprises a fluorescence resonance energy transferacceptor fluorescent dye.

Embodiment 309. The compound of one of embodiments 304 to 305, whereinthe detectable dye comprises a fluorescence resonance energy transferdonor and acceptor fluorescent dye pair connected by a linker.

Embodiment 310. The compound of one of embodiments 304 to 305, whereinthe detectable dye comprises a fluorescence resonance energy transferdonor and acceptor fluorescent dye pair connected by a linker andseparated by from 0.1 nm to 10 nm.

Embodiment 311. The compound of one of embodiments 304 to 305, whereinthe detectable dye is

Embodiment 312. The compound of one of embodiments 304 to 311 having theformula:

Embodiment 313. A method for sequencing a nucleic acid, comprising:

incorporating in series with a nucleic acid polymerase, within areaction vessel, one of four different labeled nucleotide analogues intoa primer to create an extension strand, wherein said primer ishybridized to said nucleic acid and wherein each of the four differentlabeled nucleotide analogues comprise a unique detectable label;

(ii) detecting said unique detectable label of each incorporatednucleotide analogue, so as to thereby identify each incorporatednucleotide analogue in said extension strand, thereby sequencing thenucleic acid; and

wherein each of said four different labeled nucleotide analogues are ofthe structure of one of embodiments 1 to 28, 31 to 33, 37 to 44, 50 to163, 183 to 213, 216 to 218, and 222 to 273, wherein

in the first of said four different labeled nucleotide analogues, B is athymidine or uridine hybridizing base;

in the second of said four different labeled nucleotide analogues, B isan adenosine hybridizing base;

in the third of said four different labeled nucleotide analogues, B isan guanosine hybridizing base; and

in the fourth of said four different labeled nucleotide analogues, B isan cytosine hybridizing base.

Embodiment 314. The method of embodiment 313, further comprising, aftereach of said incorporating steps, adding to said reaction vessel fourdifferent unlabeled nucleotide analogues, wherein each of said fourdifferent unlabeled nucleotide analogues are of the structure of one ofembodiments 257 to 279, wherein

in the first of said four different unlabeled nucleotide analogues, B isa thymidine or uridine hybridizing base:

in the second of said four different unlabeled nucleotide analogues, Bis an adenosine hybridizing base;

in the third of said four different unlabeled nucleotide analogues, B isa guanosine hybridizing base; and

in the fourth of said four different unlabeled nucleotide analogues, Bis a cytosine hybridizing base.

Embodiment 315. The method of one of embodiments 313 or 314, wherein atleast one of said four different labeled nucleotide analogues is anorthogonally cleavable labeled nucleotide analogue comprising acleavable moiety, said orthogonally cleavable labeled nucleotideanalogue having the structure of one of embodiments 1 to 27, 31 to 33,37 to 44, 50 to 114, 183 to 212, 216 to 218, and 222 to 246, and whereinthe method further comprises, after each of said incorporating steps,adding to said reaction vessel a cleaving reagent capable of cleavingthe cleavable moiety.

Embodiment 316. A method for sequencing a nucleic acid, comprising:

incorporating in series with a nucleic acid polymerase, within areaction vessel, one of four different nucleotide analogues into aprimer to create an extension strand, wherein said primer is hybridizedto said nucleic acid and wherein three of the four different nucleotideanalogues are different labeled nucleotide analogues each comprising aunique detectable label and one of the four different nucleotideanalogues is a different unlabeled nucleotide analogue;

detecting the presence or absence of said unique detectable label ofeach incorporated nucleotide analogue, so as to thereby identify eachincorporated nucleotide analogue in said extension strand, therebysequencing the nucleic acid; and

wherein each of said four different labeled nucleotide analogues are ofthe structure of one of embodiments 1 to 28, 31 to 33, 37 to 44, 50 to163, 183 to 213, 216 to 218, and 222 to 273, wherein

in the first of said four different labeled nucleotide analogues, B is athymidinc or uridine hybridizing base;

in the second of said four different labeled nucleotide analogues, B isan adenosine hybridizing base;

in the third of said four different labeled nucleotide analogues, B is aguanosine hybridizing base; and

in the fourth of said four different labeled nucleotide analogues, B isa cytosine hybridizing base.

Embodiment 317. The method of embodiment 316, further comprising, aftereach of said incorporating steps, adding to said reaction vessel fourdifferent unlabeled nucleotide analogues, wherein each of said fourdifferent unlabeled nucleotide analogues are of the structure of one ofembodiments 274 to 295, wherein

in the first of said four different unlabeled nucleotide analogues, B isa thymidine or uridine hybridizing base;

in the second of said four different unlabeled nucleotide analogues, Bis an adenosine hybridizing base;

in the third of said four different unlabeled nucleotide analogues, B isa guanosine hybridizing base; and

in the fourth of said four different unlabeled nucleotide analogues, Bis a cytosine hybridizing base.

Embodiment 318. The method of one of embodiments 316 or 317, wherein atleast one of said three different labeled nucleotide analogues is anorthogonally cleavable labeled nucleotide analogue comprising acleavable moiety, said orthogonally cleavable labeled nucleotideanalogue having the structure of one of embodiments 1 to 27, 31 to 33,37 to 44, 50 to 114, 183 to 212, 216 to 218, and 222 to 246, and whereinthe method further comprises, after each of said incorporating steps,adding to said reaction vessel a cleaving reagent capable of cleavingthe cleavable moiety.

Embodiment 319. A method of incorporating a nucleotide analogue into aprimer, the method comprising combining a polymerase, a primerhybridized to nucleic acid template and a nucleotide analogue within areaction vessel and allowing said polymerase to incorporate saidnucleotide analogue into said primer thereby forming an extended primer,wherein said nucleotide analogue is of the structure of one ofembodiments 1 to 163 and 183 to 273.

Embodiment 320. The method of embodiment 319, wherein L² is a cleavablemoiety and R⁵ is a detectable label, said method further comprising,after said incorporating, cleaving said cleavable moiety with a cleavingreagent.

Embodiment 321. The method of embodiment 319, wherein R⁵ is anchormoiety, said method further comprising, after said incorporating,labeling said nucleotide analog with a detectable label.

Embodiment 322. The method of embodiment 321, wherein R⁵ is an affinityanchor moiety.

Embodiment 323. The method of embodiment 322, wherein said labelingcomprises adding to the reaction vessel a compound having the formulaR¹²-L⁴-R¹³, wherein

R¹² is a complementary affinity anchor moiety binder;

R¹³ is a detectable label; and

L⁴ is a covalent linker.

Embodiment 324. The method of embodiment 321, wherein R⁵ is a chemicallyreactive anchor moiety.

Embodiment 325. The method of embodiment 324, wherein said labelingcomprises adding to the reaction vessel a compound having the formulaR^(12z)-L^(4z)-R¹³, wherein

R^(12z) is a complementary anchor moiety reactive group;

R¹² is a detectable label; and

L^(4z) is a covalent linker.

Embodiment 326. The method of embodiment 325, wherein R^(12z)-L^(4z)-R¹³has the structure of one of embodiments 296 to 321.

Embodiment 327. The method of embodiment 325, wherein L^(4z) is acleavable linker.

Embodiment 328. The method of embodiment 327, further comprising, aftersaid incorporating, cleaving said cleavable moiety with a cleavingreagent.

Embodiment 329. The method of one of embodiments 319-328, furthercomprising, after said incorporating, adding to said reaction vessel anunlabeled nucleotide analogue comprising a 3′-polymerase-compatiblecleavable moiety.

Embodiment 330. The method of one of embodiments 319-329, wherein saidmethod forms part of a sequencing by synthesis method.

Embodiment 331. A method for sequencing a nucleic acid comprising:

contacting anucleic acid having a primer hybridized to a portionthereof, with a polymerase and a first type of nucleotide analogue underconditions permitting the nucleotide polymerase to catalyzeincorporation of the nucleotide analogue into the primer if thenucleotide analogue is complementary to a nucleotide residue of thenucleic acid that is immediately 5′ to a nucleotide residue of thenucleic acid hybridized to the 3′ terminal nucleotide residue of theprimer, so as to form a nucleic acid extension product, wherein thenucleotide analogue has the structure:

wherein,

B is a nucleobase; L³ is a cleavable linker having the structure:

wherein L² is a linker;

R⁶ is a polymerase-compatible cleavable dithio moiety that when bound tothe 3′-O of the nucleotide analogues prevents a polymerase fromcatalyzing a polymerase reaction with the 3′-O of the nucleotideanalogue, wherein R6 has the structure:

Wherein R^(8A) and R^(8B) are independently hydrogen, —CH₃, —CX₃, —CHX₂,—CH₂X, —CN, -Ph, substituted or unsubstituted C₁-C₆ alkyl, substitutedor unsubstituted 2 to 6 membered heteroalkyl, substituted orunsubstituted C₃-C₆ cycloalkyl, substituted or unsubstituted 3 to 6membered heterocycloalkyl, substituted or unsubstituted phenyl, orsubstituted or unsubstituted 5 to 6 membered heteroaryl, wherein X is ahalogen;

wherein R⁹, R¹⁰ , and R¹¹ are each independently hydrogen, —CX₃, —CHX₂,—CH₂X, —OCH₃, —SCH₃, —NHCH₃, —CN, -Ph, substituted or unsubstitutedC₁-C₆ alkyl, substituted or unsubstituted 2 to 6 membered heteroalkyl,substituted or unsubstituted C₃-C₆ cycloalkyl, substituted orunsubstituted 3 to 6 membered heterocycloalkyl, substituted orunsubstituted phenyl, or substituted or unsubstituted 5 to 6 memberedheteroary, wherein X is a halogen;

R³ is an anchor moiety, wherein the identity of the anchor moiety ispredetermined and correlated to the identity of the nucleobase;

removing any nucleotide analogue not incorporated into the primer instep a);

contacting the nucleic acid in step a) with at least one compound havingthe formula R¹²-L⁴-R¹³, wherein R¹² is a complementary anchor moietybinder that rapidly reacts with the anchor moiety, thereby forming aconjugate with the anchor moiety, L⁴ is a covalent or a non-covalentlinker, and R¹³ is a detectable label;

detecting the presence of the detectable label so as to therebydetermine whether the nucleotide analogue of step a) was incorporated soas to thereby determine the identity of the complementary nucleotideresidue in the template DNA, and

wherein if the base of the nucleotide analogue a) is not complementaryto the nucleotide residue of the nucleic acid which is immediately 5′ tothe nucleotide residue of the single-stranded DNA hybridized to the 3′terminal nucleotide residue of the primer, then iteratively repeatingsteps a) through c) with a second, third, and then fourth type ofnucleotide analogue, wherein each different type of nucleotide analoguehas a different base from each other type of nucleotide analogue, untilthe nucleotide analogue has a base that is complementary.

contacting the nucleic acid with a cleaving agent, so as to (i) cleavethe cleavable linker attached to the nucleobase, and (ii) cleave thecleavable dithio moiety, thereby resulting in a 3′-OH on the growing DNAstrand; and

iteratively performing steps a) through e) for each nucleotide residueof the nucleic acid to be sequenced so as to thereby determine thesequence of the nucleic acid.

Embodiment 332.The embodiment of 331 wherein the first, second, third,and fourth type of nucleotide analogue have different anchor moieties,and wherein each different anchor moiety is complementary to a differentanchor moiety binder containing a detectable label.

Embodiment 333The method of any one of embodiments 331-332, wherein thedifferent binding molecules each have a different detectable label.

Embodiment 334. The method of any one of claims 332-333, wherein duringor subsequent to step a) the nucleic acid is contacted with a nucleotideanalogue having the structure:

-   -   B is a nucleobase, which is complementary to the same nucleobase        as is the nucleotide analogue of step a);    -   R^(8A) and R^(8B) are independently hydrogen, —CH₃,        —CX_(3;)—CHX₂, —CH₂X, —CN, -Ph, substituted or unsubstituted        C₁-C₆ alkyl, substituted or unsubstituted 2 to 6 membered        heteroalkyl, substituted or unsubstituted C₃-C₆ cycloalkyl,        substituted or unsubstituted 3 to 6 membered heterocycloalkyl,        substituted or unsubstituted phenyl, or substituted or        unsubstituted 5 to 6 membered heteroaryl, wherein X is a        halogen;    -   R⁹, R¹⁰, and R¹¹ are each independently hydrogen, —CX₃, —CHX₂,        —CH₂X, —OCH₃, —SCH₃, —NHCH₃, —CN, -Ph, substituted or        unsubstituted C₁-C₆ alkyl, substituted or unsubstituted 2 to 6        membered heteroalkyl, substituted or unsubstituted C₃-C₆        cycloalkyl, substituted or unsubstituted 3 to 6 membered        heterocycloalkyl, substituted or unsubstituted phenyl, or        substituted or unsubstituted 5 to 6 membered heteroaryl, wherein        X is a halogen.

Embodiment 335. A method for sequencing a nucleic acid comprising:

a) contacting a nucleic acid having a primer hybridized to a portionthereof, with a polymerase and a first, second, third, and fourth typeof nucleotide analogue under conditions permitting the polymerase tocatalyze incorporation of the nucleotide analogue into the primer if thenucleotide analogue is complementary to a nucleotide residue of thenucleic acid that is immediately 5′ to a nucleotide residue of thenucleic acid hybridized to the 3′ terminal nucleotide residue of theprimer, so as to form a DNA extension product, wherein each type ofnucleotide analogue has the structure:

wherein, L³ is a cleavable linker having the structure:

wherein L² is a linker;

wherein B is a nucleobase, wherein the base of each type of nucleotideanalogue is independently different from the base of the remaining threetypes of nucleotide analogue, wherein one base is a thymidine or uridinehybridizing base, one base is an adenosine hybridizing base, one base isa guanosine hybridizing base, and one base is a cytosine hybridizingbase;

R⁶ is a polymerase-compatible cleavable dithio moiety that when bound tothe 3′-O prevents a nucleotide polymerase from catalyzing a polymerasereaction with the 3′-O of the nucleotide analogue, wherein R⁶ has thestructure:

-   -   wherein R^(8A) and R^(8B) are independently hydrogen, CH₃, —CX₃,        —CHX₂, —CH₂X, —CN, -Ph, substituted or unsubstituted C₁-C₆        alkyl, substituted or unsubstituted 2 to 6 membered heteroalkyl,        substituted or unsubstituted C₃-C₆ cycloalkyl, substituted or        unsubstituted 3 to 6 membered heterocycloalkyl, substituted or        unsubstituted phenyl, or substituted or unsubstituted 5 to 6        membered heteroaryl, wherein X is a halogen;    -   wherein R⁹, R¹⁰, and R¹¹ are eachindependently hydrogen, —CX₃,        —CHX₂, —CH₂X, —OCH₃, —SCH₃, —NHCH₃, —CN, -Ph, substituted or        unsubstituted C₁-C₆ alkyl, substituted or unsubstituted 2 to 6        membered heteroalkyl, substituted or unsubstituted C₃-C₆        cycloalkyl, substituted or unsubstituted 3 to 6 membered        heterocycloalkyl, substituted or unsubstituted phenyl, or        substituted or unsubstituted;    -   R⁵ is an anchor moiety, wherein the identity of the anchor        moiety is predetermined and correlated to the identity of the        nucleobase, and wherein the anchor moiety of each type of        nucleotide analogue is complementary to a different anchor        binding moiety from each of the remaining nucleotide analogues;

b) contacting the nucleic acid in step a) with at least a first, second,third, and fourth type of compound having the formula R¹²-L⁴-R¹³,wherein R¹² is a complementary anchor moiety binder that rapidly reactswith an anchor moiety, thereby forming a conjugate with the anchormoiety, wherein the anchor moiety binder of each type of compound iscomplementary to the anchor moiety of one type of nucleotide analoguefrom step a), wherein L⁴ is a covalent linker, and R¹³ is a detectablelabel, wherein each type of compound has a different detectable label,that is correlated to the identity of the anchor moiety binder;

c) determining the identity of the detectable label bound to thenucleotide analogue incorporated in step a) so as to thereby determinethe identity of the incorporated nucleotide analogue,

d) contacting the nucleic acid with a cleaving agent, so as to (i)cleave the cleavable linker attached to the nucleobase, and (ii) cleavethe cleavable dithio moiety, thereby resulting in a 3′—OH in the growingDNA strand; and

e) iteratively performing steps a) through d) for each nucleotideresidue of the nucleic acid to be sequenced so as to thereby determinethe sequence of the nucleic acid.

Embodiment 336. A method for sequencing a nucleic acid comprising:

a) contacting a nucleic acid having a primer hybridized to a portionthereof, with a nucleotide polymerase and a first, second, third, andfourth type of nucleotide analogue under conditions permitting thenucleotide polymerase to catalyze incorporation of the nucleotideanalogue into the primer if the nucleotide analogue is complementary toa nucleotide residue of the nucleic acid that is immediately 5′ to anucleotide residue of the nucleic acid hybridized to the 3′ terminalnucleotide residue of the primer, so as to form a nucleotide extensionproduct, wherein each type of nucleotide analogue has the structure:

wherein, L³ is a cleavable linker having the structure:

wherein L² is a linker;

wherein B is a nucleobase, wherein the base of each type of nucleotideanalogue is independently different from the base of the remaining threetypes of nucleotide analogue, wherein one base is a thymidinc or uridinehybridizing base, one base is an adenosine hybridizing base, one base isa guanosine hybridizing base, and one base is a cytosine hybridizingbase:

R⁶ is a polymerase-compatible cleavable dithio moiety that when bound tothe 3′-O prevents a nucleotide polymerase from catalyzing a polymerasereaction with the 3′-O of the nucleotide analogue, wherein R⁶ has thestructure:

whereinR^(8A) and R^(8B) are independently hydrogen, CH₃, —CX₃, —CHX₂,—CN, -Ph, substituted or unsubstituted C₁-C₆ alkyl, substituted orunsubstituted 2 to 6 membered heteroalkyl, substituted or unsubstitutedC₃-C₆ cycloalkyl, substituted or unsubstituted 3 to 6 memberedheterocycloalkyl, substituted or unsubstituted phenyl, or substituted orunsubstituted 5 to 6 membered heteroaryl, wherein X is a halogen;

wherein R⁹, and ^(Rn) are eachindependently hydrogen, —CX₃, —OCH₃,—SCH₃, —NHCH₃, —CN, -Ph, substituted or unsubstituted C₁-C₆ alkyl,substituted or unsubstituted 2 to 6 membered heteroalkyl, substituted orunsubstituted C₃-C₆ cycloalkyl, substituted or unsubstituted 3 to 6membered heterocycloalkyl, substituted or unsubstituted phenyl, orsubstituted or unsubstituted;

wherein for the first and second type of nucleotide analogue R⁵ is ananchor moiety, wherein the identity of the anchor moiety ispredetermined and correlated to the identity of the nucleobase, andwherein the anchor moiety of each type of nucleotide analogue iscomplementary to a different anchor binding moiety from the remainingnucleotide analogue;

wherein for the third and fourth type of nucleotide analogue R5 is adetectable label, wherein the detectable label for the third type ofnucleotide analogue is different from the fourth type;

b) removing any non-incorporated nucleotide analogue;

c) detecting the presence of either of the detectable label of the thirdor fourth type of nucleotide analogue incorporated in step a) so as tothereby determine the identity of the incorporated nucleotide and theidentity of the complementary residue in the nucleic acid; wherein ifthe base of the third and fourth type of nucleotide analogue is notcomplementary, contacting the nucleic acid in step a) with at least afirst, and second type of compound having the formula R¹²-L⁴-R¹³,wherein R¹² is a complementary anchor moiety binder that rapidly reactswith an anchor moiety, thereby forming a conjugate with the anchormoiety, wherein the anchor moiety binder of first type of compound iscomplementary to the anchor moiety of first type of nucleotide analogueand the anchor moiety binder of second type of compound is complementaryto the anchor moiety of second type of nucleotide analogue, wherein L⁴is a covalent linker, and R¹¹ is a detectable label, wherein the labelon the first type of compound and second type of compound are different;

d) determining the identity of the detectable label bound to thenucleotide analogue incorporated in step a) so as to thereby determinethe identity of the incorporated nucleotide analogue,

e) contacting the nucleic acid with a cleaving agent, so as to (i)cleave the cleavable linker attached to the nucleobase, and (ii) cleavethe cleavable dithio moiety, thereby resulting in a 3′—OH; and

f) iteratively performing steps a) through e) for each nucleotideresidue of the nucleic acid to be sequenced so as to thereby determinethe sequence of the nucleic acid.

Embodiment 337. A method for sequencing a nucleic acid comprising:

a) contacting a nucleic acid having a primer hybridized to a portionthereof, with a nucleotide polymerase and a first, second, third, andfourth type of nucleotide analogue under conditions permitting thenucleotide polymerase to catalyze incorporation of the nucleotideanalogue into the primer if the nucleotide analogue is complementary toa nucleotide residue of the nucleic acid that is immediately 5′ to anucleotide residue of the nucleic acid hybridized to the 3′ terminalnucleotide residue of the primer, so as to form a nucleotide extensionproduct, wherein the first, second, and third type of nucleotideanalogue each have the structure:

wherein, B is a nucleobase, wherein L³ is a cleavable linker having thestructure:

wherein L² is a linker;

wherein R⁵ is an anchor moiety, wherein the identity of the anchormoiety is predetermined and correlated to the identity of thenucleobase, and wherein the anchor moiety of each type of nucleotideanalogue is complementary to a different anchor binding moiety from theremaining nucleotide analogues; wherein the fourth type of nucleotideanalogue has the structure:

wherein B is a nucleobase;

wherein the base of each type of nucleotide analogue is independentlydifferent from the base of the remaining three types of nucleotideanalogue, wherein one base is a thymidine or uridine hybridizing base,one base is an adenosine hybridizing base, one base is a guanosinehybridizing base, and one base is a cytosine hybridizing base;

wherein R⁶ for each of the first, second, third, and fourth types ofnucleotide analogue is a polymerase-compatible cleavable dithio moietythat when bound to the 3′-O prevents a nucleotide polymerase fromcatalyzing a polymerase reaction with the 3′-O of the nucleotideanalogue, wherein R⁶ has the structure:

whereinR^(8A) and Ras _(are) i_(n)d_(epen)d_(e)ntl_(y) h_(y)drogen, CH₃,—CX₃, —CHX₂, -MX, —CN, -Ph, substituted or unsubstituted C₁-C₆ alkyl,substituted or unsubstituted 2 to 6 membered heteroalkyl, substituted orunsubstituted C₃-C₆ cycloalkyl, substituted or unsubstituted 3 to 6membered heterocycloalkyl, substituted or unsubstituted phenyl, orsubstituted or unsubstituted 5 to 6 membered heteroaryl, wherein X is ahalogen; wherein R⁹, le , and ^(Rii) are eachindependently hydrogen,—CX₃, —CHX2, —CH₂X, —OCH₃, —SCH₃, —NHCH₃, —CN, -Ph, substituted orunsubstituted C₁-C₆ alkyl, substituted or unsubstituted 2 to 6 memberedheteroalkyl, substituted or unsubstituted C₃-C₆ cycloalkyl, substitutedor unsubstituted 3 to 6 membered heterocycloalkyl, substituted orunsubstituted phenyl, or substituted or unsubstituted;

b) removing any non-incorporated nucleotide analogue;

c) contacting the nucleic acid with a first, second, and third type ofcompound having the formula R¹²-L⁴R¹³,

-   -   wherein R¹² is a complementary anchor moiety binder that rapidly        reacts with an anchor moiety, thereby forming a conjugate with        the anchor moiety,    -   wherein the anchor moiety binder of the first type of compound        is complementary to the anchor moiety of first type of        nucleotide analogue, the anchor moiety binder of second type of        compound is complementary to the anchor moiety of second type of        nucleotide analogue, and the anchor moiety binder of the third        type of compound is complementary to the anchor moiety of the        third type of nucleotide analogue, wherein L⁴ is a cleavable        linker,        -   wherein L⁴of each type of compound is different;

d) removing any unincorporated nucleotides or unconjugated anchor moietybinders and detecting whether there is an absence of detectable labelbound to the incorporated nucleotide analogue of step a), therebydetermining whether the incorporated nucleotide was of the fourth typeof nucleotide analogue;

e) if a detectable label is detected in step d), contacting the nucleicacid with a means of cleaving L⁴of the first type of compound, whereinthe means does not cleave L³ or R⁶ of any type of nucleotide analogue,and the means does not cleave L⁴ of the second and third type ofcompound;

f) removing any unconjugated anchor moiety binders and detecting whetherthere is an absence of detectable label bound to the incorporatednucleotide of step a), thereby determining that identity of theincorporated nucleotide is of the first type of nucleotide analogue;

g) if a detectable label is detected in step f), contacting the nucleicacid with a means of cleaving L⁴ of the second type of compound, whereinthe means does not cleave L³ or R⁶ of any type of nucleotide analogue,and the means does not cleave L⁴ of the third type of compound;

h) removing any unconjugated anchor moiety binders and detecting whetherthere is an absence of detectable label bound to the incorporatednucleotide of step a), thereby determining that identity of theincorporated nucleotide is of the second type of nucleotide analogue;contacting the nucleic acid with a cleaving agent, so as to (i) cleavethe cleavable linker attached to the nucleobase, and (ii) cleave thecleavable dithio moiety, thereby resulting in a 3′-OH; and

j) iteratively performing steps a) through i) for each nucleotideresidue of the nucleic acid to be sequenced so as to thereby determinethe sequence of the nucleic acid.

Embodiment 338. The method of any one of embodiments 333-337, whereinduring or subsequent to step a) the nucleic acid is contacted with fourdifferent types of nucleotide analogue, each having the structure:

wherein B is a nucleobase, wherein the base of each type of nucleotideanalogue is independently different from the base of the remaining threetypes of nucleotide analogue, and wherein one base is a thymidine oruridine hybridizing base, one base is an adenosine hybridizing base, onebase is a guanosine hybridizing base, and one base is a cytosinehybridizing base;

wherein R^(8A) and R^(8B) are independently hydrogen, —CH₃, —CX₃, —CHX₂,—CH₂X, —CN, -Ph, substituted or unsubstituted C₁-C₆ alkyl, substitutedor unsubstituted 2 to 6 membered heteroalkyl, substituted orunsubstituted C₃-C₆ cycloalkyl, substituted or unsubstituted 3 to 6membered heterocycloalkyl, substituted or unsubstituted phenyl, orsubstituted or unsubstituted 5 to 6 membered heteroaryl, wherein X is ahalogen; and

wherein R⁹, ^(Ric))^(,) and R^(il) are eachindependently hydrogen, —CX₃,—CHX₂, —CH₂X, —OCH₃, —SCH₃, —NHCH₃, —CN, -Ph, substituted orunsubstituted C₁-C₆ alkyl, substituted or unsubstituted 2 to 6 memberedheteroalkyl, substituted or unsubstituted C₃-C₆ cycloalkyl, substitutedor unsubstituted 3 to 6 membered heterocycloalkyl, substituted orunsubstituted phenyl, or substituted or unsubstituted 5 to 6 memberedheteroary, wherein X is a halogen.

Embodiment 339. The method of any one of embodiments 332-338, whereinthe anchor moieties and labeling moieties are selected from the groupcomprising:

Embodiment 340. The method of any one of embodiments 332-339, whereinthe complementary anchor binding moiety is selected from the groupconsisting of:

a streptavidin moiety, or

Embodiment 341. The method of any one of embodiments 332-340, wherein L³has the structure:

Embodiment 342. A method of synthesizing a base-attached cleavablelinker having the structure:

comprising: 313

a) reacting 3-trimethylsilanyl-prop-2-yn-1-ol with DMSO, acetic acid andacetic anhydride to provide Trimethyl(3-((methylthio)methoxy)prop-1-yn-1-yl) silane;

b) contacting Trimethyl (3-((methylthio)methoxy)prop-1-yn-1-yl)silanewith Sulfuryl chloride/cyclohexene followed by reaction with potassiumthiotosylate to produceS-(((3-(trimethylsilyl)prop-2-yn-1-yl)oxy)methyl)4-methylbenzenesulfonothioate;

c) contacting S-4(3-(trimethylsilypprop-2-yn-1-ypoxy)methyl)4-mothylbenzcnesulfonothioatc with2,2,2-trifluoro-N-(2-mercapto-2-methylpropyl) acetamide in the presenceof triethylamine, followed by;

d) contacting with tetrabutylammonium fluoride to form2,2,2-trifluoro-N-(2-methyl-2-(((prop-2-yn-1-yloxy)methyl)disulfanyl)propyl)acetamide.

e) reacting 5′-O-tBDMS-3′-O-polymerase compatible cleavable blockinggroup-5(7)-Iodo-nucleoside with2,2,2-trifluoro-N-(2-methyl-2-(((prop-2-yn-1-yloxy)methyl)disulfanyl)propyl)acetamidein the presence of Pd(0), CuI, triethylamine which results in theformation of 5′-O-tBDMS-3′-O-polymerase compatible cleavable blockinggroup-2′-deoxynucleoside-5(7)-2,2,2-trifluoro-5-ypethynyl)oxy)methyl)disulfanyl)-2-methylpropyl)acetamide.

f) reacting the above product from step e) with tetrabutylammoniumfluoride to remove 5′-O-tBDMS group permitting the formation of3′-O-polymerase compatible cleavable blocking group-2′-deoxynucleoside-5(7)-2,2,2-trifluoro-5-ypethynyl)oxy)methyl)disulfanyl)-2-methylpropyl)acetamide;

g) contacting the 3′-O-polymerase compatible cleavable blockinggroup-2′-deoxynucleoside-5(7)-2,2,2-trifluoro-5-yl)ethynyl)oxy)methyl)disulfanyl)-2-methylpropyl)acetamideproduced in step f) with 2-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one,tetrabutylammonium pyrophosphate, tributylamine, I₂, pyridine, and NH₄OHunder condition permitting the formation of 3′-O-polymerase compatiblecleavable blockinggroup-5(7)-(3-(((1-amino-2-methylpropan-2-yl)disulfanyl)methoxy)prop-1-yn-1-yl)-2′-deoxynucleo side-5′-triphosphate

EXAMPLES Example 1 Design and Synthesis of Novel Disulfide Linker basedNucleotides as Reversible Terminators for DNA Sequencing by Synthesis

We first demonstrated that the four 3′-O-alkyldithiomethyl-dNTPs(3′-O-DTM NRTs) (FIGS. 30A-30D) can extend a primer in a single baseextension reaction and terminates its further extension. Using these 4nucleotide analogues as substrates, we then performed a complete 4-stepSBS reaction. We used MALDI-TOF MS analysis to evaluate the results ofpolymerase extension by a single 3′-O-DTM-dNTP into the growing DNAstrand (FIGS. 30B and 30D). Due to the small size of the DTM label, the3′-O-DTM-dNTPs are efficient substrates for the DNA polymerase. In theSBS cycles, the natural nucleotides are restored after eachincorporation and cleavage, producing a growing DNA strand that bears nomodifications and will not impede further polymerase reactions.

We have also carried out similar single base extension and terminationreactions using the chemically cleavable fluorescent nucleotidereversible terminator 3′-O-DTM-dCTP-SS-BodipyFL. After single baseextension and cleavage of the DTM moiety from the 3′-O and between thebase and fluorophore, the resulted extended primer can be furtherextended with the same nucleotide, 3′-O-DTMACTP-SS-Bodip/FL (FIG. 46 ).These results clearly indicate that multiple NRTs, 3′-O-DTM-dNTP-SS-Dye,can be incorporated and cleaved simultaneously resulting in long readsequencing.

Continuous Polymerase Extension Using 3′-O-Et-SS-dNTPs (3′-O-DTM-dNTPs)and Characterization by MALDI-TOF Mass Spectrometry. To verify that the3′-O-DTM-dNTPs are incorporated accurately in a base-specific manner inthe polymerase reaction, four consecutive DNA extension and cleavagereactions were carried out in solution with 3′-O-DTM-dNTPs assubstrates. This allowed the isolation of the DNA product at each stepfor detailed molecular structure characterization as shown in FIG. 30B.

We performed a complete consecutive 4-step SBS reaction that involvedincorporation of each complementary 3′-O-DTM-dNTPs, followed byMALDI-TOF MS analysis for sequence determination, and cleavage of the3′-O-DTM blocking group from the DNA extension product to yield a free3′—OH group for incorporating the next nucleotide analogue. Atemplate-primer combination was designed in which the next fournucleotides to be added were A, C, G and T. As shown in FIG. 30B, theSBS reaction was initiated with the 13-mer primer annealed to a DNAtemplate. When the first complementary nucleotide, 3′-O-Et-SS-dATP(3′-O-DTM-dATP), was used in the polymerase reaction, it wasincorporated into the primer to form a DNA extension product with amolecular weight of 4387 Dalton as confirmed by MALDI-TOF MS with theappearance of a single peak (FIG. 30B (a)Top left). These resultsindicated that the 3′-O-DTMdATP was quantitatively incorporated into the13-mer DNA primer. After THP treatment to remove the DTM group from theDNA product and HPLC purification, the cleavage was confirmed by thepresence of a single MS peak at 4268 Da, corresponding to the DNAproduct with the 3′-O-DTM group removed (FIG. 30B (b)Top right). Thenewly formed DNA extension product with a free 3′—OH group was then usedin a second polymerase reaction to incorporate a 3′-O-Et-SS-dCTP(3′-O-DTM-dCTP) which gave a single MS peak at 4675 Da (FIG. 30B (c),indicating incorporation of a 3′-O-DTM-dCTP into the growing DNA strandin this cycle. After THP treatment, a single MS peak of the cleavage DNAproduct appeared at 4551 Da (FIG. 30B (d), which demonstrated thecomplete removal of the DTM group from the DNA extension product. FIG.30B (left) shows the third incorporation of 3′-O-Et-SS-dGTP(3′-O-DTM-dGTP) into this growing DNA strand. The accurate masses of thecorresponding DNA products were obtained by MALDI-TOF MS for the thirdnucleotide incorporation (5002 Da, FIG. 30B (e), and cleavage reaction(4887 Da, FIG. 30B, f). Finally, 3′-O-Et-SS-dTTP (3′-O-DTM-dTTP)incorporation in the fourth cycle and a final removal of the DTM groupby THP was verified, as appropriate masses for the corresponding DNAproducts were obtained by MALDI-TOF MS for the fourth nucleotideincorporation (5301 Da, FIG. 30B,(g) and cleavage reaction (5194 Da,FIG. 30B (h). These results demonstrate that all four 3′-O-DTM-dNTPs areefficiently incorporated base-specifically as reversible terminatorsinto the growing DNA strand in a continuous polymerase reaction, andthat the 3′-OH capping group on the DNA extension products isquantitatively cleaved by THP.

Polymerase Extension reaction Using 3′-O-tBu-SS-dCTP-SS-BodipyFL(3′-O-DTM-dCTPSS-BodipyFL) and Characterization by MALDI-TOF MassSpectrometry (FIG. 46 ). Polymerase extension reactions consisted of 20pmol of a synthetic DNA template, 60 pmol of primer(5′-TCTCTGGCCGCGTGTCT-3′) (SEQ ID NO:3), 100 pmol of a single nucleotidereversible terminator (3′-O-tBu-SS-dCTP-SS-Bodipy), 1× ThennoPol IIreaction buffer (New England Biolabs, MA), 2 units Therminator II DNApolymerase, 2 nmol MnCl₂, and deionized H₂O in a total volume of 20 μL.Reactions were conducted in an ABI GeneAmp PCR System 9700 with initialincubation at 94° C. for 20 sec, followed by 38 cycles of 80° C. for 20sec, 45° C. for 30 sec, and 65° C. for 90 sec. After the extensionreaction, the extension product was precipitated by ethanol and pelletdissolved in 10 μL deionized water. A fraction of extension reaction wasdesalted using a C18 ZipTip column (Millipore, Mass.) and analyzed byMALDI-TOF MS (ABI Voyager, DE). The remaining large fraction treatedwith THP (tris-(hydroxypropyl)phosphine) at a final concentration of 5mM and incubated at 65° C. for 3 minutes to regenerate the 3′—OH groupin preparation for the next extension reaction. A small fraction wasdesalted as before and analyzed by MALDI-TOF MS. Remaining fraction ofextended primer was used for second round of extension reaction (FIG. 46).

Sequencing by Synthesis Reactions using 3′-O-DTM-dNTPs. Polymeraseextension reactions consisted of 20 pmol of a synthetic 51-mer DNAtemplate (5′GAGGCCAAGTACGGCGGGTACGTCCTTGACAATGTGTACATCAACATCACC-3′) (SEQID NO:9), 60 pmol of primer (5′-CACATTGTCAAGG-3′) (SEQ ID NO:2) or apreviously extended and THP cleaved DNA product, 100 pmol of a single3′-O-DTM nucleotide reversible terminator (3′-ODTM-dATP, 3′-O-DTM-dCTP,3′-O-DTM-dGTP, or 3′-O-DTM-dTTP), 1× ThermoPol reaction buffer (NewEngland Biolabs, MA), 2 unit Therminator™ III DNA polymerase anddeionized H₂O in a total volume of 20 μL. Reactions were conducted in athermal cycler (MJ Research, MA). After initial incubation at 94° C. for20 sec, the reaction was performed for 36 cycles at 80° C. for 20 sec,45° C. for 40 sec and 65° C. for 90 sec.

After the extension reaction, a small aliquot of the reaction productwas desalted using a C18 ZipTip column (Millipore, Mass.) and analyzedby MALDT-TOF MS (ABT Voyager, DE). The remaining product wasconcentrated under vacuum and purified by reverse phase HPLC on anXTerra MS C18, 2.5 μm 4.6 mm×50 mm column (Waters, Mass.) to obtain thepure extension product. Mobile phase: A, 8.6 mM triethylamine/100 mM1,1,1,3,3,3-hexafluoro-2-propanol in water (pH 8.1); B, methanol.Elution was performed at 40° C. with a 0.5 mL/min flow rate, and from88% A/12% B to 65.5% A/34.5% B linear gradient for 90 min. The purifiedproduct was used in the subsequent polymerase extension reaction.

Cleavage reactions to remove the 3′-O-DTM group from the DNA extensionproducts with THP to regenerate the 3′—OH group were carried out bydissolving 100 pmol extension products in 10 μL of 5 mMTris(2-hydroxypropyl)phosphine (THP) solution (pH 9.0), and incubatingat 65° C. for 3 min. Following dilution in 1 mL deionized H₂O anddesalting in an Amicon Ultra-0.5 centrifugal filter unit with Ultracel-3membrane (Millipore), 2 μL of the resulting solution was used to obtainthe MALDI-TOF mass spectrum. After further reverse phase HPLC as above,each cleavage product was used as primer in the subsequent polymeraseextension reaction. Four consecutive nucleotide additions are shown inFIGS. 30A-30D.

Synthesis of 3′-O-ethyldithiome thyl-2′-deoxynucleoside-5′-triphosphates (3 ′-O-DTMdNTPs): Synthesis of3′-O-ethyldithiomethyl-dTTP (7a) FIG. 47 ):

3′-O-methylthiomethyl-5′-O-tent-butyldimethylsilyl thymidine (2a): To astirring solution of the 5′-O-tert-butyldimethylsilyl thymidine (la,1.07 g, 3 mmol) in DMSO (10 mL) was added acetic acid (2.6 mL, 45 mmol)and acetic anhydride (8.6 mL, 90 mmol). The reaction mixture was stirredat room temperature until the reaction was complete (48 h), which wasmonitored by TLC.

Then the mixture was added slowly to a saturated solution of sodiumbicarbonate under vigorous stirring and extracted with ethyl acetate(3×30 mL). The combined organic layers were dried over Na₂SO₄ andfiltered. The filtrate was concentrated to dryness under reducedpressure and the compound was purified by silica gel columnchromatography (ethyl acetate/hexane: 1:2) to give pure product 2a (0.97g, 74%). ¹H NMR (400 MHz, CDCl₃) δ: 8.16 (s, 1H), 7.48 (s, 1H), 6.28 (m,1H), 4.62 (m, 2H), 4.46 (m, 1H), 4.10 (m, 1H), 3.78-3.90 (m, 2H), 2.39(m, 1H), 2.14 (s, 3H), 1.97 (m, 1H), 1.92 (s, 3H), 0.93 (s, 9H). 0.13(s. 3H); HRMS (FAB′) calc'd for C₁₈H₃₃N₂O₅SSi [(M+H)+]: 417.1879, found:417.1890.

3 ′-O-ethyldithiomethyl-5′-O-tert-butyldimethylsilyl thymidine (5a)3′-O-methylthiomethyl5′-O-tent-butyldimethylsilyl thymidine (2a, 453 mg,1.09 mmol) was dissolved in anhydrous dichloromethane (20 mL), followedby addition of triethylamine (0.18 mL, 1.31 mmol, 1.2 eq.) and molecularsieve (3 A, 2 g). The mixture was cooled in an ice-bath after stirringat room temperature for 30 min and then a solution of sulfuryl chloride(redistilled, 0.1 mL, 1.31 mmol, 1.2 eq.) in anhydrous dichloromethane(3 mL) was added dropwise over 2 minutes. The ice-bath was removed andthe reaction mixture was stirred further for 30 min. Then potassiumptoluenethiosulfonate (375 mg, 1.65 mmol, 1.5 eq.) in anhydrous DMF (2mL) was added to the mixture. Stirring was continued at room temperaturefor additional hour followed by addition of ethanethiol (0.17 mL, 2.2mmol, 2 eq.). The reaction mixture was stirred at room temperature for30 min and quickly filtered through celite. The filter was washed withdichloromethane and the organic fraction was concentrated. The residuewas purified by Flash column chromatography (ethyl acetate/hexane: 2:1)to give pure product 5a (261 mg, 52%).¹H NMR (400 MHz, CDCl₃) δ: 8.66(br. s, 1H), 7.49 (s, IH), 6.30 (dd, J=7.2, 11.2 Hz, IH), 4.83 (dd,J=15.2, 37.2 Hz, 2H), 4.49 (d, J=8.0 Hz, 1H), 4.14 (d, J=3.2 Hz, 1H),3.80 (m, 2H), 2.77 (dd, J=10.0, 19.6 Hz, 2H), 2.47(m, 1H), 2.03 (m, 1H),1.93 (s, 3H), 1.35 (t, J=8.8 Hz, 2H), 0.95 (s, 9H), 0.14 (s, 6H). ¹³CNMR (75 MHz, CDCl₃): δ 164.00, 150.59, 135.61, 111.35, 85.33, 79.76,77.98, 77.81, 63.89, 38.10, 33.64, 26.33, 18.74, 14.84,12.89,-4.85,-5.03.

3′-O-ethyldithiomethyl thymidine (3′-O-DTM-T, 6a):3′-O-ethyldithiomethyl-5′-O-tert-butyldimethylsilyl thymidine (5a, 240mg, 0.52 mmol) was dissolved in anhydrous THF (10 mL) and a THF solutionof tetrabutylammonium fluoride (1.0M, 1.04 mL , 1.04 mmol, 1.5 eq.) wasadded. The reaction mixture was stirred at room temperature for 4 hours.The reaction mixture was concentrated in vacuo, saturated NaHCO₃solution (50 mL) was added and the mixture was extracted withdichloromethane (3×20 mL). The organic layer was dried over anhydrousNa₂SO₄, filtered, concentrated and the obtained crude mixture waspurified by flash column chromatography (dichloromethane/methanol: 20/1)to give 3′-O-ethyldithiomethyl thymidine 6a (119 mg, 66%). 1H NMR (300MHz, CDCl₃) δ: 7.44 (s, 1H), 6.15 (t, J=8.8 Hz, 1H), 4.83 (dd, J=11.4.23.4 Hz, 2H), 4.46 (m, 1H), 4.12 (m, 2H), 3.80 (m, 2H), 2.77 (dd, J=7.5,14.7 Hz, 2H), 2.34(m, 2H), 2.04 (s, 1H),1.90 (s, 3H), 1.34 (t, J=7.5 Hz,3H). ¹³C NMR (75 MHz. CDCl₃): δ 164.37,150.88, 137.26, 111.53, 87.20,85.29, 78.52, 62.82, 37.49, 33.59, 14.85, 12.89. HRMS (ESI+) calc'd forC13H20N2O5S₂Na [(M+Na)+]: 371.0711, found: 371.0716.

3′-O-ethyldithiomethyl-dTTP (3′-O-DTM-TTP 7a): 3′-O-ethyldithiomethylthymidine (6a, 50 mg, 0.14 mmol), tetrabutylammonium pyrophosphate (197mg, 0.36 mmol, 2.5 eq.) and2-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one (44 mg, 0.22 mmol, 1.5 eq)were dried separately overnight under high vacuum at ambienttemperature. The tetrabutylammonium pyrophosphate was dissolved indimethylformamide (DMF, 1 mL) under argon followed by addition oftributylamine (1 mL). This mixture was injected into the solution of2-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one in (DMF, 2 mL) underargon. After stirring for 1 h, the reaction mixture was added to thesolution of 3′-O-ethyldithiomethyl thymidine and stirred further for 1hour at room temperature. Iodine solution (0.02 M iodine/pyridine/water)was then injected into the reaction mixture until a permanent browncolor was observed. After 10 min, water (30 mL) was added and thereaction mixture was stirred at room temperature for additional 2 hours.The resulting solution was extracted with ethyl acetate (2×30 mL). Theaqueous layer was concentrated in vacuo to approximately 20 mL, andtransferred to two centrifuge tubes (50 mL). brine (1.5 mL) and absoluteethanol (35 mL) were added to each tube, followed by vigorous shakingAfter being placed at −80° C. for 2 h, the tube was centrifuged (10 minat 4200 rpm) to afford the crude product as a white precipitate. Thesupernatant was poured out, the white precipitate was diluted with 5 mlof water and purified by ion exchange chromatography on DEAE-SephadexA-25 at 4° C. using a gradient of TEAB (pH 8.0; 0.1-1.0 M). The crudeproduct was further purified by reverse-phase HPLC to afford 7a. HRMS(ESI—) calc'd for C₁₃H₂₂N₂O₁₄S₂P₃[(M−H)⁻]: 586.9725, found: 586.9727.³¹P-NMR (121.4MHz, D₂O): δ−10.83 (s, 1P), −10.98 (s, 1P), −20.53 (t,J=21Hz, 1P).

N²-Dimethylformamidino-2′-deoxyguanosine (2b): To a suspension of2′-deoxyguanosine (lb, 1.33 g, 5 mmol) in dry DMF (20 mL) was added N,N-dimethylformamide dimethyl acetal (1.5 mL, 11 mmol) and the reactionmixture was stirred at room temperature overnight. The solvent wasremoved and the residue triturated with methanol and filtered. The solidwas washed with methanol to give a white solid 2b (90%, 1.44 g). ¹H NMR(400 MHz, DMSO-d₆) δ 11.28 (s, 1H), 8.57 (s, 1H), 8.04 (s, 1H), 6.26(dd, J=7.9, 6.1 Hz, 1H), 5.30 (d, J=3.8 Hz, 1H), 4.93 (t, J=5.5 Hz, 1H),4.40 (dt, J=5.8, 2.8 Hz, 1H), 3.85 (td, J=4.5, 2.5 Hz, 1H), 3.56 (m,2H), 3.17 (s, 3H), 3.04 (s, 3H), 2.60 (m, 1H), 2.25 (m, 1H).

Dimethylformamidino-5′-O-DMT-2′-deoxyguanosine (3b):N²-DMF-2′-deoxyguanosine (2b, 1.38 g, 4.3 mmol, 1 eq.) was dissolved inanhydrous pyridine (30 mL), and 4, 4′-dimethoxytrityl chloride (1.74 g,5.2 mmol, 1.2 eq.) was added. After stirring at room temperature for 4hours, the reaction mixture was poured into saturated sodium bicarbonatesolution (200 mL) and the precipitate was collected by suctionfiltration, washed with water and hexane. The obtained crude produce waspurified by silica gel column chromatography (dichloromethane/methanol:30/1) to give N²-DMF-5′-O-DMT-2′deoxyguanosine 3b (1.84 g, 69%) as awhite solid. ¹H NMR (400 MHz, CDCl₃) δ9.13 (s, 1H), 8.57 (s, 1H), 7.71(s, 1H), 7.3 (m, 2H), 7.34-7.20 (m, 6H), 7.18 (t, J=2.8 Hz, 1H),6.90-6.72 (m, 4H), 6.40 (t, J=6.6 Hz, 1H), 4.64(m, 1H), 4.15 (m, 1H).3.81 (m, 1H), 3.78 (m, 6H), 3.43 (dd, J=10.1. 4.8 Hz, 1H), 3.32 (dd,J=10.1, 5.0 Hz, 1H), 3.11 (s, 3H), 3.06 (s, 3H), 2.65-2.48 (m, 2H).

N²-Dimethylformamidino-3′-O-methylthiomethyl-5′-O-DMT-2′-deoxyguanosine(4b): To a stirred solution of the N²-DMF-5′-O-DMT-2′-deoxyguanosine(1.33 g, 2.1 mmol) in DMSO (10 mL) was added acetic acid (2.1 mL, 36mmol) and acetic anhydride (5.4 mL, 56 mmol). The reaction mixture wasstirred at room temperature until the reaction was complete (24 h),which was monitored by TLC. Then the mixture was added slowly to asolution of sodium bicarbonate under vigorous stirring and extractedwith ethyl acetate (3×30 mL). The combined organic layers were driedover Na₂SO₄ and filtered. The filtrate was concentrated to dryness underreduced pressure and the desired compound was purified by silica gelcolumn chromatography (ethyl acetate/hexane: 1/2) to give pure product4b (1.27 g, 88%) as a white solid. ¹H NMR (400 MHz, CDCl₃) 6 9.73 (s,1H), 8.58 (s, 1H), 7.73 (s, 1H), 7.47-7.38 (m, 2H), 7.37-7.17 (m, 7H),6.87-6.77 (m, 4H), 6.33 (dd, J=7.7, 6.1 Hz, 1H), 4.72-4.63 (m, 3H),4.25-4.18 (m, 1H), 3.80 (s, 6H), 3.34 (m, 2H), 3.14 (s, 3H), 3.09 (s,3H), 2.64-2.48 (m, 2H), 2.13 (s, 3H): ¹³C NMR (75 MHz, CDCl₃): δ158.96,158.69, 158.50, 150.61, 144.88, 136.19, 136.02, 130.41, 128.49, 128.33,127.35, 120.85, 113.61, 86.96, 84.19, 83.64, 74.01, 64.05, 55.65, 41.74,38.31, 35.61, 14.26.

N²-Dimethylformamidino-3′-O-ethyldithiomethyl-5′-O-DMT-2′-deoxyguanosine(7b): N²-DMF-3′-O-methylthiomethyl-5′-O-DMT-2′-deoxyguanosine (684 mg,1.0 mmol) was dissolved in anhydrous dichloromethane (20 mL), followedby addition of triethylamine (0.17 mL, 1.2 mmol, 1.2 eq.) and molecularsieve (3 Å, 2 g). The mixture was cooled in an ice-bath after stirringat room temperature for 30 min and then a solution of sulfuryl chloride(0.095 mL, 1.2 mmol, 1.2 eq.) in anhydrous dichloromethane (3 mL) wasadded dropwise over 2 minutes. The ice-bath was removed and the reactionmixture was stirred further for 30 min. Then potassium4-toluenethiosulfonate (341 mg, 1.5 mmol, 1.5 eq.) in anhydrous DMF (2mL) was added to the mixture. Stirring was continued at room temperaturefor an additional hour followed by addition of ethanethiol (0.16 mL, 2.0mmol, 2 eq.). The reaction mixture was stirred at room temperature for30 min and quickly filtered through celite. The filter was washed withdichloromethane and the organic fraction was concentrated. The residuewas purified by silica gel column chromatography (ethyl acetate/hexane:2/1) to give pure product 7b (255 mg. 35%). ¹H NMR (400 MHz, CDCl₃) 69.55 (s, 1H), 8.58 (s, 1H), 7.73 (s, 1H), 7.47-7.38 (m, 2H), 7.37-7.27(m, 6H), 7.27-7.18 (m, 1H), 6.88-6.79 (m, 4H), 6.34 (t, J=7.0 Hz, 1H),4.86 (s, 2H),4.65 (m, 1H), 4.25 (m, 1H), 3.80 (d, J=0.9 Hz, 6H),3.44-3.28 (m, 2H), 3.16-3.07 (s, 3H), 3.10 (s, 3H), 2.75 (qd, J=7.4, 0.7Hz, 2H), 2.62-2.54 (m, 2H), 1.29 (t, J=13.5, 4H). ¹³C NMR (75 MHz,CDCl₃): δ 158.99, 158.50, 157.30, 150.57, 144.84, 136.06, 135.95,130.41, 128.47, 128.36, 127.38, 120.88, 113.65, 87.04, 84.12, 83.61,79.68, 78.48, 64.02, 55.65, 41.74, 38.34, 35.60, 33.60, 14.87, 14.59.

3′-O-ethyldithiomethyl-2′-deoxyguanosine (8b): The mixture ofN²-DMF-3′-ethyldithiomethyl-5¹-O-DMT-2′-deoxyguanosine (280 mg, 0.38mmol), ammonium hydroxide (10 mL) and methanol (10 mL) was stirred atroom temperature until the reaction was complete (4 h), which wasmonitored by TLC. After evaporation of the solvent under reducedpressure, the crude solid was treated with 3% trichloroacetic acidsolution in dichloromethane for 10 min. Then the mixture was addedslowly to the solution of sodium bicarbonate under vigorous stirring andextracted with ethyl acetate (3 ×30 mL). The combined organic layerswere dried over Na₂SO₄ and filtered. The filtrate was concentrated todryness under reduced pressure and the desired compound was purified bysilica gel column chromatography (dichloromethane/methanol: 20/1) togive 3′-ethyldithiomethyl-2′deoxyguanosine 8b (72 mg, 51%). ¹H NMR (300MHz, DMSO-d₆) δ10.61 (s, 1H), 7.93 (s, 1H), 6.45 (bs, 2H), 6.07 (dd,J=8.5, 5.7 Hz, 1H), 5.06 (bs, 1H), 4.95 (s, 2H), 4.51 (d, J=5.3 Hz, 1H),3.99 (m, 1H), 3.55 (d, J=4.3 Hz, 2H), 2.80 (q, J=7.3 Hz, 2H), 2.72-2.56(m, 1H), 2.43-2.39 (m, 1H), 1.28 (t, J=7.3 Hz, 3H). HRMS (ESL) calc'dfor C₁₃H₁₉N₅O₄S₂Na [(M+Na)⁺]: 396.0776, found: 396.0770.

3′-O-ethyldithiomethyl-dGTP (9b): The preparation procedure was similarto the synthesis of 7a. 3′-ethyldithiomethyl-2′deoxyguanosine (8b, 64mg, 0.17 mmol), tetrabutylammonium pyrophosphate (238 mg, 0.44 mmol, 2.5eq.) and 2-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one (53 mg, 0.27mmol, 1.5 eq) were dried separately over night under high vacuum atambient temperature in three round bottom flasks. The tetrabutylammoniumpyrophosphate was dissolved in dimethylformamide (DMF, 1 mL) under argonfollowed by addition of tributylamine (1 mL). The mixture was injectedinto the solution of 2-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one in(DMF, 2 mL) under argon. After stirring for 1 h, the reaction mixturewas added to the solution of 3′-O-ethyldithiomethyl thymidine andstirred further for 1 hour at room temperature. Iodine solution (0.02 Miodine/pyridine/water) was then injected into the reaction mixture untila permanent brown color was observed. After 10 min, water (30 mL) wasadded and the reaction mixture was stirred at room temperature for anadditional 2 hours. The resulting solution was extracted with ethylacetate (2×30 mL). The aqueous layer was concentrated in vacuo toapproximately 20 mL, and transferred to two centrifuge tubes (50 mL).Brine (1.5 mL) and absolute ethanol (35 mL) were added to each tube,followed by vigorous shaking. After being placed at −80° C. for 2 h, thetube was centrifuged (10 min at 4200 rpm) to offer the crude product asa white precipitate. The supernatant was poured out, the whiteprecipitate was diluted with 5 ml of water and purified with anionexchange chromatography on DEAE-Sephadex A-25 at 4° C. using a gradientof TEAB (pH 8.0; 0.1-1.0 M). The crude product was further purified byreverse-phase HPLC to afford 9b.

3) Synthesis of 3′-O-ethyldithiomethyl-dATP (8c) (FIG. 48 ).N6-Benzoyl-5′-O-trityl-2′-deoxyadenosine (2c):N⁶-Benzoyl-2′-deoxyadenosine (1c, 1.07 g, 3.0 mmol, 1 eq.) was dissolvedin anhydrous pyridine (30 mL), and trityl chloride (1.00 g, 3.6 mmol,1.2 eq.) was added. After stirring at room temperature for 1 day, thereaction mixture was poured into saturated sodium bicarbonate solution(200 mL) and the precipitate was collected by suction filtration, washedwith water and hexane. The obtained crude product was purified by silicagel column chromatography (dichloromethane/methanol: 30/1) to giveN⁶-Benzoyl-5′-O-trityl-2′-deoxygadenosine 2c (1.45 g, 81%) as a whitesolid. ¹H NMR (400 MHz, CDCl₃) δ9.12 (s, 1H), 8.74 (s, 1H), 8.15 (s,1H), 8.08-8.00 (m, 2H), 7.62 (m, 1H), 7.52 (m, 2H), 7.46-7.38 (m, 6H),7.34-7.20 (in, 9H), 6.50 (t, J=6.5 Hz, 1H), 4.74 (d, J=4.7 Hz, 1H), 4.19(td, J=4.8, 3.5 Hz, 1H), 3.49-3.42 (m, 2H), 2.90 (m, 1H), 2.58 (m, 1H).

N-Benzoyl-3′-O-methylthiomethyl-5′-O-trityl-2′-deoxyadenosine (3c): To astirred solution of the N⁶-Benzoyl-5′-O-trityl-2′-deoxyadenosine (1.72g, 2.93 mmol) in DMSO (10 mL) was addcd acetic acid (2.8 mL, 48 mmol)and acetic anhydride (72 mL, 75 mmol). The reaction mixture was stirredat room temperature until the reaction was complete (24 h), which wasmonitored by TLC.

Then the mixture was added slowly to a solution of sodium bicarbonateunder vigorous stirring and extracted with ethyl acetate (3 ×30 mL). Thecombined organic layers were dried over Na₂SO₄ and filtered. Thefiltrate was concentrated to dryness under reduced pressure and thedesired compound was purified by silica gel column chromatography (ethylacetate/hexane: 1/2) to give pure product 3c (1.35 g, 71%) as a whitesolid. ¹H NMR (400 MHz, CDCl₃) δ9.07 (s, 1H), 8.74 (s, 1H), 8.19 (s,1H), 8.05 (dt, J=7.2, 1.4 Hz, 2H), 7.67-7.49 (m, 3H), 7.49-7.39 (m, 6H),7.36-7.22 (m, 9H), 6.48 (dd, J=7.6, 6.0 Hz, 1H), 4.79 (m, 1H), 4.66 (m,2H), 4.31 (td, .1 =4.8, 2.7 Hz, 1H), 3.51-3.38 (m, 2H), 2.89 (m, 1H),2.64 (m, 1H), 2.15 (s, 3H). ¹³C NMR (75 MHz, CDCl₃): δ 165.03, 153.03,151.82, 149.88, 143.87, 141.78, 134.05, 133.19, 129.27, 129.02, 128.35,128.28, 127.67, 123.83, 87.52, 85.43, 85.59, 76.85, 74.05, 63.98, 37.94,30.13, 14.27.

N⁶-Benzoyl-3′-O-ethyldithiomethyl-5′-O-trityl-2′-deoxyadenosine (6c):3′-O-methylthiomethyl-5′-O-Trityl-2′-de oxyadenosine (3c, 861 mg, 1.31mmol) was dissolved in anhydrous dichloromethane (20 mL), followed byaddition of tricthylamine (0.19 mL, 1.5 mmol, 1.2 eq.) and molecularsieve (3 A, 2 g). The mixture was cooled in an ice-bath after stirringat room temperature for 0.5 hour and then a solution of sulfurylchloride (0.11 mL, 1.5 mmol, 1.2 eq.) in anhydrous dichloromethane (3mL) was added dropwise during 2 minutes. The ice-bath was removed andthe reaction mixture was stirred further for 30 min. Then potassiumptoluenethiosulfonate (595 mg, 2.62 mmol, 1.5 eq.) in anhydrous DMF (3mL) was added to the mixture. Stirring was continued at room temperaturefor an additional hour followed by addition of ethanethiol (0.47 mL,6.55 mmol, 2 eq.). The reaction mixture was stirred at room temperaturefor 30 min and quickly filtered through celite. The filter was washedwith dichloromethane and the organic fraction was concentrated. Theresidue was purified by silica gel column chromatography (ethylacetate/hexane: 2/1) to give pure product 6c (615 mg, 67%). ¹H NMR (400MHz, CDCl₃) δ9.04 (s, 1H), 8.74 (s, 1H), 8.18 (s, 1H), 8.05 (d, J=7.2Hz, 2H), 7.67-7.59 (m, 1H), 7.59-7.50 (m, 2H), 7.50-7.38 (m, 6H),7.36-7.21 (m, 9H), 6.47 (dd, J=7.8, 5.9 Hz, 1H), 4.90 (s, 2H), 4.75 (dt,J=5.4, 2.5 Hz, 1H), 4.35 (td, J=4.9, 2.5 Hz, 1H), 3.45 (m, 2H),3.00-2.86 (m, 1H), 2.85-2.71 (m, 2H), 2.68 (m, 1H), 1.33 (t, J=7.4, 3H).

N⁶-Benzoyl-3′-O-ethyldithiomethyl-2′-deoxyadenosine (7c):N⁶-Benzoyl-3′-ethyldithiomethyl-5¹-O-trityl-2′-deoxyadenosine (6c, 381mg, 0.54 mmol) was treated with 3% trichloroacetic acid solution indichloromethane at room temperature for 10 min. Then the mixture wasadded slowly to a solution of sodium bicarbonate under vigorous stirringand extracted with ethyl acetate (3×30 mL). The combined organic layerswere dried over Na₂SO₄ and filtered. The filtrate was concentrated todryness under reduced pressure and the residue of the desired compoundwas purified by silica gel column chromatography(dichloromethane/methanol: 20/1) to give 7c (169 mg, 68%). ¹H NMR (400MHz, DMSO-d₆) δ11.18 (s, 1H), 8.77 (s, 1H), 8.71 (s, 1H), 8.10-8.02 (m,2H), 7.66 (t, J=7.6 Hz, 1H), 7.56 (t, J=7.6 Hz 2H), 6.47 (dd, J=8.0, 6.0Hz, 1H), 5.15 (t, J=5.5 Hz, 1H), 5.00 (s, 2H), 4.65 (dt, J=5.4, 2.4 Hz,1H), 4.12 (td, J=4.7, 2.2 Hz, 1H), 3.72-3.55 (m, 2H), 3.02-2.88 (m, 1H),2.84 (q, J=7.3 Hz, 2H), 2.61 (m, 1H), 1.40-1.15 (m, 3H). ¹³C NMR (75MHz, DMSO-d₆): δ 166.47, 152.83, 152.47, 151.27, 143.87, 134.22, 133.30,129.33, 126.78, 86.18, 84.79, 79.35, 78.80, 62.37, 36.93, 33.04, 15.21.

3′-O-ethyldithiomethyl-dATP (8c): Compound 7c (100 mg, 0.22 mmol) andproton sponge (60 mg, 0.28 mmol) were dried in a vacuum desiccator overP₂O₅ overnight and dissolved in trimethyl phosphate (2 ml). Freshlydistillated POCI₃ (30 μL, 0.32 mmol) was added dropwise and the mixturewas stirred for 2 h at 0° C. Tributylammonium pyrophosphate (452 mg,0.82 mmol) and tributylamine (450 uL, 1.90 mmol) in anhydrous DMF(1.9mL) was added in one portion at room temperature and the solutionstirred for additional 30 min. Triethylammonium bicarbonate solution(TEAB, 0.1 M; pH 8.0; 10 mL) was added and the mixture was stirred for 1h at room temperature. Then concentrated NH₄OH (10 mL) was added andstirring continued for 3 h at room temperature. The mixture wasconcentrated under vacuum and the crude product was purified by anionexchange chromatography on DEAE-Sephadex A-25 at 4° C. using a gradientof TEAB (pH 8.0; 0.1-1.0 M), followed by a further purification byreverse-phase HPLC to afford 8c.

Synthesis of 3′-O-ethyldithiomethyl-dCTP (3′-O-DTM-dCTP, 7d) (FIG. 49 ).N⁴-Benzoyl-3′-O-methylthiomethyl-5′-O-tert-butyldimethylsilyl-2′-deoxycytidine(2d):To a stirred solution ofN⁴-Benzoyl-5′-O-tert-butyldimethylsilyl-2′-deoxycytidine (1.5 g, 3.37mmol) in anhy DMSO (6.5 ml) was added acetic acid (2.9 ml) and aceticanhydride (9.3 ml). The mixture was stirred at room temperature for 2days, and then quenched by adding saturated NaHCO₃ solution (50 ml). Thereaction mixture was extracted with ethyl acetate (50 mL x 3) and thecombined organic layers dried over anhydrous Na₂SO₄. The crude productafter concentration was purified by flash column chromatography (ethylacetate: Hexane 8:2) to give a white powder (1.26 g, 74%). ¹H NMR (400MHz, Methanol-d₄) δ8.50 (d, J=7.5 Hz, 1H), 8.05-7.97 (m, 2H), 7.72-7.61(m, 2H), 7.61-7.52 (m, 2H), 6.23 (t, J=6.3 Hz, 1H), 4.81-4.71(m, 2H),4.58 (dt, J=6.4, 3.3 Hz, 1H), 4.24 (q, J=3.1 Hz, 1H), 4.02 (dd, J=11.5,3.3 Hz, 1H), 3.91 (dd, J=11.5, 2.8 Hz, 1H), 2.75-2.59 (m, 1H), 2.24 (dt,J=13.9, 6.3 Hz, 1H), 2.18 (s, 3H), 0.98 (s, 9H), 0.19 (d, J=3.3 Hz, 6H).HRMS (APCI⁺) calc'd for C₂₄H₃₅N₃O₅SSi [(M+H)⁺]: 506.2145, found:506.2124.

N⁴-Benzoyl-3′-O-ethyldithiomethyl-5′-O-tert-butyldimethylsilyl-2′-deoxycytidine(5d):To a stirred solution of 2d (612 mg, 1.21 mmol) in anhydrousdichloromethane (10 ml), triethylamine (168 μL, 1.21 mmol) and 4Amolecular sieve (1 g) were added. The reaction mixture was stirred atroom temperature for 30 minutes and then cooled in an ice-bath. SO₂Cl₂(98 μL, 1.21 mmol) dissolved in anhydrous dichloromethane (5 ml) wasadded dropwise to the mixture. Then the ice bath was removed, and thereaction mixture was stirred for at room temperature for 30 minutes.Potassium p-toluenethiosulfonate (425 mg, 1.9 mmol) dissolved inanhydrous DMF (6254) was added into the reaction mixture, and afterbeing stirred for additional 30 minutes, ethancthiol (174 μL, 2.4 mmol)was added and stirring continued at room temperature for an additional30 minutes. The reaction mixture was filtered, concentrated, and thenextracted with saturated sodium bicarbonate and dichloromethane (3×50mL). The organic phase was dried over Na₂SO₄, concentrated, and purifiedby flash column chromatography using a gradient of ethyl acetate- hexanefrom 5:5(v/v) to 8:2(v/v), yielding 563.2 mg (84%) white foam. ¹H NMR(400 MHz, Methanol-d₄) δ8.55-8.42 (m, 1H), 8.00 (dt, J=8.4, 1.1 Hz, 2H),7.70-7.45 (m, 4H), 6.23 (q, J=6.9, 6.4 Hz, 1H), 5.01-4.88 (m, 2H), 4.56(ft, J=6.5, 3.1 Hz, 1H), 4.30-4.19 (m, 1H), 4.00 (m, J=11.4, 3.2, 0.8Hz, 1H), 3.94-3.76 (m, 1H), 2.81 (qd, J=7.3, 0.9 Hz, 2H), 2.76-2.68 (m,1H), 2.31-2.17 (m, 1H), 1.40-1.25 (m, 3H), 1.00-0.85 (m, 9H), 0.21-0.03(m, 6H). HRMS (APCI⁺) calc'd for C₂₅H₃₇N₃O₅S₂Si [(M+Na)⁺]: 574.1841,found: 574.1826.

N⁴-Benzoyl-3′-O-ethyldithiomethyl-2′-deoxycytidine (6d): To a stirredsolution of 5d (526 mg, 0.95 mmol) in a mixture of tetrahydrofuran (3ml) and methanol (9 ml), NH₄F (1.8 g) powder was added in small portionsand stirred at room temperature for 3 days. The crude product wasconcentrated and purified by flash column chromatography using agradient of ethyl acetate- Hexane from 2:8(v/v) to 7:3(v/v), affording awhite solid powder (233 mg, 56%). ¹H NMR (400 MHz, Methanol-d₄) 1H NMR(400 MHz, Methanol-d₄) δ8.54 (d, J=7.5 Hz, 1H), 8.04-7.97 (m, 2H),7.71-7.43 (m, 4H), 6.25 (t, 1H), 5.01-4.89 (m, 2H), 4.56 (dt, J=6.0, 3.0Hz, 1H), 4.23 (q, J=3.4 Hz, 1H), 3.92-3.76 (m, 2H), 2.84 (q, J=7.3 Hz,2H), 2.71 (m, J=13.9, 5.9, 2.9 Hz, 1H), 2.31-2.19 (m, 1H), 1.36 (t,J=7.3 Hz, 3H).HRMS (AP co calc'd for C₁₉H₂₃N₃O₅S₂ [(M+H)⁺]: 438.1157,found: 438.1136.

3′-O-ethyldithiomethyl)-dCTP (7d): Compound 6d (60 mg, 0.14 mmol) andproton sponge (40 mg, 0.19 mmol) were dried in a vacuum desiccator overP₂O₅ overnight, dissolved in trimethyl phosphate (1 ml) and cooled in anicebath. Freshly distillated POCl₃ (19 μL, 0.2 mmol) was added dropwiseand stirred for 2 h at 0° C. Tributylammonium pyrophosphate (255 mg,0.47 mmol) and tributylamine (27.6 uL, 0.12 mmol) in anhydrous DMF (1.5mL) was added in one portion at room temperature followed by anadditional stirring for 30 min. Triethylammonium bicarbonate solution(TEAB) (0.1 M; pH 8.0; 7.5 mL) was added and the mixture was stirred for1 h at room temperature. Then concentrated NH₄OH (7.5 mL) was added andstirring continued overnight at room temperature. The mixture wasconcentrated under vacuum and the crude product was purified by anionexchange chromatography on DEAE-Sephadex A-25 at 4° C. using a gradientof TEAB (pH 8.0; 0.1-1.0 M), followed by a further purification byreverse-phase HPLC to afford 7d.

Synthesis ofAminopropynyl-3′-O-t-butyldithiomethyl-2′-Deoxynucleoside-5′Triphosphates(PA-3′-O-DTM-dNTPs, FIG. 50 ).5-(3-aminopropynyl)-3′-O-t-butyldithiomethyl-dCTP (5-PA-3′-O-DTM-dCTP).

N⁴-DMF-5-Iodo-2′-deoxycytidine (2): A mixture of 5-iodo-2′-deoxycytidine(1, 1.25 g, 3.5 mmol) and NN-dimethylformamide dimethyl acetal (1.25 mL,9.1 mmol) in dry DMF (20 mL) was stirred at room temperature overnight.After this period, the solvent was removed and the residue trituratedwith methanol and filtered. The solid was washed with methanol to give awhite solid 2 (88% , 1.25 g). ¹H NMR (400 MHz, DMSO-d₆) δ8.57 (s, 1H),8.46 (s, 1H), 6.10 (t, J=6.4 Hz, 1H), 5.21 (d, J=4.3 Hz, 1H), 5.11 (t,J=5.0 Hz, 1H), 4.24 (p, J=4.8, 4.1 Hz, 1H), 3.83 (q, J=3.4 Hz, 1H),3.71-3.53 (m, 2H), 3.21 (s, 3H), 3.13 (s, 3H), 2.21 (dt, J=13.7, 5.0 Hz,1H), 2.04 (dt, J=12.9, 6.3 Hz, 1H).

N⁴-DMF-5-Iodo-5¹-O-Trityl-2′-deoxycytidine (3):N⁴-DMF-5-iodo-2′-deoxycytidine (2, 0.93 g, 2.3 mmol, 1 eq.) wasdissolved in anhydrous pyridine (30 mL), and trityl chloride (0.78 g,2.8 mmol, 1.2 eq.) was added. After stirring at room temperature for 1day, the reaction mixture was poured into saturated sodium bicarbonatesolution (200 mL) and the precipitate was collected by filtration,washed with water and hexane. The obtained crude product was purified bycolumn chromatography (dichloromethane/methanol: 30/1) to giveN⁴-DMF-5-iodo-5′-O-Trityl-2′-deoxycytidine 3 (1.12 g, 75%) as a whitesolid. ¹H NMR (400 MHz, DMSO-d6) 6 8.59 (s, 1H), 8.13 (s, 1H), 7.45 (m,6H), 7.30 (m, 6H), 7.26 (m, 3H), 6.13 (t, J=6.0 Hz, 1H), 5.29 (d, J=4.5Hz, 1H), 4.23 (td, J=6.8, 5.5, 3.2 Hz, 1H), 3.94 (m, 1H), 3.28-3.18 (m,5H), 3.14 (d, J=0.8 Hz, 3H), 2.28 (ddd, J=13.3, 6.0, 3.3 Hz, 1H),2.18-2.06 (m, 1H).

N⁴-DMF-5-[3-(trifluoroacetamido)propynyl]-5′-O-trityl-2′-deoxycytidine(4): Under nitrogen, a mixture ofN⁴-DMF-5-iodo-5′-O-Trityl-2′-deoxycytidine (244 mg, 0.375 mmol, 1.2eq.), CuI (20 mg, 0.11 mmol) and Triethylamine (0.15 mL) in dry DMF (5mL) was stirred at room temperature for 5 min followed by the additionof N-propargyl trifluoroacetamide (0.2 g, 1.36 mmol), and Pd(PPh₃)4 (50mg, 0.04 mmol). After stirring at room temperature in the darkovernight, the reaction mixture was added dropwise into brine (200 mL)under vigorous stirring and the precipitate was collected by suctionfiltration, and washed with water and hexane. The obtained crude producewas purified by column chromatography (100% ethyl acetate followed bydichloromethane/methanol: 30/1) to giveN⁴-DMF-5[3-(trifluoroacetamido)propynyl]-5′-O-trityl-2′- deoxycytidine 4(199 mg, 79%) as alight yellowbrown solid. ¹H NMR (400 MHz, DMSO-d₆)δ9.97 (t, J=5.5 Hz, 1H), 8.62 (s, 1H), 8.03 (s, 1H), 0.45 (m, 6H), 7.30(m, 6H), 7.26 (m, 3H), 6.14 (t, J=6.6 Hz, 1H), 5.32 (d, J=4.5 Hz, 1H),4.26 (dq, J=7.8, 3.8 Hz, 1H), 4.06 (d, J=5.5 Hz, 2H), 4.04-3.94 (m, 1H),3.29 (m, 1H), 3.20 (s, 3H), 3.16 (m, 1H), 3.09 (s, 3H), 2.29 (m, 1H),2.14 (m, 1H).

N⁴-DMF-5-[3-(trifluoroacetamido)propynyl]-5′-O-trityl-3′-O-methylthiomethyl-2′deoxycytidine(5): To a solution of theN⁴-DMF-5-[3-(trifluoroacetamido)propynyl]-5′-O-Trityl-2′-deoxycytidine(4, 1.47 g, 2.19 mmol) in DMSO (10 mL) with stirring was added aceticacid (2.3 mL, 39 mmol) and acetic anhydride (6.1 mL, 64 mmol). Thereaction mixture was stirred at room temperature until the reaction wascomplete (24 h), which was monitored by TLC. Then the reaction mixturewas added to a solution of sodium bicarbonate under vigorous stirring ,the precipitate was collected by suction filtration, and washed withwater and hexane. The obtained crude product was purified by columnchromatography dichloromethane/methanol: 30/1) to give pure product 5(1.22 g, 77%) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ8.81 (s, 1H),8.47 (s, 1H), 7.48 (m, 6H), 7.34 (m, 6H), 7.25(m, 3H), 6.31 (t, J=6.3Hz, 1H), 6.24 (s, 1H), 4.66 (m, 1H), 4.61 (m, 2H), 4.16 (m, 1H), 3.90(m, 2H), 3.54 (dd, J=10.8, 2.9 Hz, 1H), 3.29 (dd, J=10.8, 3.4 Hz, 1H),3.18 (d, J=4.5 Hz, 6H), 2.70 (m, J=13.9, 6.2, 3.9 Hz, 1H), 2.25 (dt,J=13.8, 6.4 Hz, 1H), 2.05 (s, 3H).

N⁴-DMF-5-[3-(trifluoroacetamido)propynyl]-5′-O-trityl-3′-O-(tert-butyldithiomethyl)-2′deoxycytidine(7):N⁴-DMF-5-[3-(trifluoroacetamido)propynyl]-5′-O-trityl-3′-O-methylthiomethyl-2′deoxycytidine(5, 1.05 g, 1.74 mmol) was dissolved in anhydrous dichloromethane (20mL), followed by addition of triethylamine (0.3 mL) and molecular sieve(3 Å, 2 g). The mixture was cooled in an ice-bath after stirring at roomtemperature for 0.5 hour and then a solution of sulfuryl chloride (0.16mL) in anhydrous dichloromethane (3 mL) was added dropwise during 2minutes. The ice-bath was removed and the reaction mixture was stirredfurther for 30 min. Then potassium p-toluenethiosulfonate (620 mg) inanhydrous DMF (3 mL) was added to the mixture. Stirring was continued atroom temperature for additional 1 hour followed by addition of tertbutylmercaptan (0.38 mL,). The reaction mixture was stirred at roomtemperature for 0.5 hour and quickly filtered through celite. The filterwas washed with dichloromethane and the organic fraction wasconcentrated. The residue was purified by silica gel columnchromatography (dichloromethane/methanol: 30/1) to give crude 7.

5-[3-(trifluoroacetamido)propynyl]-3′-O-(tert-butyldithiomethyl)-2′-deoxycytidine(8): CrudeN⁴-DMF-5-[3-(trifluoroacetamido)propynyl]-5′-O-Trity-3′-O-(tert-butyldithiometh1)-2′deoxycytidine7 was dissolved in dichloromethane and treated with 3% trichloroaceticacid solution at room temperature for 10 min. The mixture was addedslowly to a saturated solution of sodium bicarbonate under stirring andextracted with ethyl acetate (3×30 mL). The combined organic layers weredried over Na₂SO₄ and filtered. The filtrate was concentrated to drynessunder reduced pressure and the residue of the desired compound waspurified by column chromatography (dichloromethane/methanol: 30/1) togive 8 (206 mg, 23% from 5). ¹H NMR (400 MHz, CDCl₃) δ 8.88 (bs, 1H),8.77 (s, 1H), 8.17 (bs, 1H), 8.03 (s, 1H), 6.20 (bs, 1H), 6.12 (t, J=6.4Hz, 1H), 4.80 (m, 2H), 4.52(m, 1H), 4.32 (m, 2H), 4.17 (d, J=2.4 Hz,1H), 3.97-3.83 (m, 2H), 2.52 (m, 1H), 2.25 (m, 1H), 1.30 (s, 9H). HRMS(Fab⁺) calc'd for C₁₉H₂₆F₃N₄O₅S₂[(M+H)⁺]: 511.1297, found: 511.1288.

5-(3-trifluoroacetamidopropynyl)-3′-O-tert-butyldithiomethyl-dCTP (9):5-[3-(trifluoroacctamido)propynyl]-3′-O-(tert-butyldithiomethl)-2′-deoxycytidine(8, 70 mg, 0.14 mmol), 2-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one (44mg, 0.22 mmol) and tetrabutylammonium pyrophosphate (197 mg, 0.36 mmol)were dried separately overnight under high vacuum. Under argon, thetetrabutylammonium pyrophosphate was dissolved in DMF (1 mL) followed byaddition of tributylamine (1 mL). The mixture was injected into thesolution of 2-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one in DMF (2 mL).After stirring for 1 h, the reaction mixture was added to the solutionof 3′-O-tert-butyldithiomethyl thymidine and stirred further for 1 hourat room temperature. Iodine solution (0.02 M iodine/pyridine/water) wasthen added to the reaction mixture until a permanent brown color wasobserved. After 10 min, water (30 mL) was added and the reaction mixturewas stirred at room temperature for an additional 2 hours. The resultingsolution was extracted with ethyl acetate (2×30 mL). The aqueous layerwas concentrated in vacuo to approximately 20 mL, and transferred to twocentrifuge tubes (50 mL). Brine (1.5 mL) and absolute ethanol (35 mL)were added to each tube, followed by vigorous shaking. After beingplaced at −80° C. for 2 h, the tube was centrifuged (10 min at 4200 rpm)to afford the crude product as a white precipitate, which was dilutedwith 5 mL of water and 5 mL ammonium hydroxide. The reaction mixture wasstirred at room temperature overnight. After evaporation of the solventunder reduced pressure, the mixture was purified by anion exchangechromatography on DEAE-Sephadex A-25 at 4° C. using a gradient of TEAB(pH 8.0; 0.1-1.0 M). The crude product was further purified byreverse-phase HPLC to afford 9.

The synthesis of other three nucleotides5-(3-aminopropynyl)-3′-O--tert-butyldithiomethyl-dTTP,7-(3-aminopropynyl)-3′-O-tert-butyldithiomethyl-7-deaza-2′-dATP, and7-(3-aminopropynyl)3′-O-tert-butyldithiomethyl-7-deaza-2′-dGTP followsthe same procedure as reported above.

Synthesis of Linker, attachment of Dye to the linker and couplingreaction with PA-3′-ODTM-dNTPs to make 3′-O and base protected3′-O-DTM-dNTPs-SS-Dye terminators (FIG. 43 and FIG. 45 ).

3-(methylthio)methoxy)propanenitrile (2). To a stirred solution of3-hydroxypropanenitrile (1, 3 g, 42.3 mmol) in dry DMSO (78 ml), aceticacid (36 ml) and acetic anhydride (120 ml) were added.

The mixture was stirred at room temperature for 2 days, and thenquenched by adding to a saturated NaHCO₃ solution (150 ml). The aqueoussolution was extracted with ethyl acetate (150 mL×3) and the combinedorganic phase was dried over anhydrous Na₂SO₄. The crude product 2 wasconcentrated and purified by flash column chromatography (ethylacetate:hexane 8:2). Light yellow oil (2.41 g, 44%) was afforded. ¹H NMR(400 MHz, Chloroform-d) δ4.69 (s, 2H), 3.77 (t, J=6.2 Hz, 2H), 2.65 (t,J=6.3 Hz, 2H), 2.19 (s, 3H).

Fmoc-NH-PEG₄-SH (5). Fmoc-NH-PEG₄-NHS ester (200 mg, 0.34 mmole) wasdissolved in 4 ml DCM, then 2-aminoethanethio-HCl (38.8 mg, 0.37 mmole)and DIPEA (0.24m1, 1.36 mmole) were added. The reaction mixture wasstirred at r.t. for 4 h. Then the solvent was evaporated under reducedpressure. The product was purified using a silica gel column(DCM/Methanol, 10/1). Removal of solvent afforded compound 5 as acolorless syrup. MALDI-TOF MS found: 548; Cal: 546.6.

S-(2-cyanoethoxy)methyl) 4-methylbenzenesulphonothioate (3) and Compound6. To a stirred solution of 2 (50 mg, 0.38 mmol) in anhydrousdichloromethane (3.75 ml), cyclohexene (195 μL, 1.92 mmol) was added.The reaction mixture was cooled in an ice-bath. SO₂Cl₂ (34.5 uL, 0.42mmol) were added dropwise to the mixture. Then the ice bath was removed,and the reaction mixture was stirred at room temperature for 30 min.Potassium p-toluenethiosulfonate (87 mg, 0.38 mmol) dissolved inanhydrous DMF (3 mL) was added into the reaction mixture to afford 3,and after stirring for an additional hour, Fmoc-NH-PEG₄-SH (5, 197 mg,2.4 mmol) in anhydrous dichloromethane(0.5 ml) was added, followed byadditional stirring at room temperature for lh. The reaction mixture wasconcentrated, and to the residue DCM (2 ml) was added to dissolve thesyrup, and 15 times (volume) of diethyl ether was added at 0° C.allowing formation of precipitate. Centrifugation was used to collectthe precipitate giving the crude product 6. HPLC (C18 column, elutiongradient:B from 10% in A to 80% in 40 min, A : 0.1% TFA in water, B:Acetonitrile) was used to purify the product, removal of solvent offeredpure product 6 as a colorless syrup. MALDI-TOF MS found: 663; Cal:661.6.

Hydrolysis of 6 to compound 7. Compound 6 (20 mg) was dissolved in 600ul acetonitrile, 200 ul of TEA was added and the reaction mixture wasshaken at r.t. for 16 h. Then the solvent and TEA were evaporated todryness and 100 ul dichloromethane (DCM) was added to dissolve theresidue. Diethyl ether (1.2 ml) was added to the DCM solution allowingformation of precipitate. Centrifugation was used to spin down theprecipitate and the supernatant was discarded. This DCM---dissolvingethyl ether precipitation process was repeated 3 times. Thorough removalof the solvent gave product 7 as a white solid. MALDI--TOF MS found:442, Cal: 439

Bodipy addition (8). Compound 7 (8 mg) was dissolved in DMF (0.2 ml) andBodipy NHS ester (5 mg, dissolved in 300 ul Methanol) was added. Afteraddition of lul TEA, the reaction mixture was shaken for 4 h. Thesolvent was removed by evaporation and the product was purified by HPLC(C-18 reverse phase column, elution gradient: B 10% in A to 80% in 40min, A: 0.1% TFA in water, B: Acetonitrile). The fraction containingproduct was collected, combined and dried, yielding product 8, MALDI-TOFMS found: 714, Cal: 714.2

Hydrolysis of CN to COOH (9). Compound 8 (4 mg) was dissolved in a mixedsolution of PBS buffer(100 mM, pH 7.5) and methanol (400 ul/100 ul),then 0.5 mg of nitrilase (dissolved in 10 ul PBS) was added.The reactionmixture was shaken in a 37° C. incubator for 24 h, and another 0.5 mgnitrilase was added. The reaction mixture was kept in the incubator for3 days. HPLC was used to purify the final product 9 (C-18 reverse phasecolumn, elution gradient: B 0% in A to 50% in 40 min, A: HFIP-TEAbuffer, B: Methanol).TOF---MALDI MS found (M+1): 735; Cal: 733.2.

3′-O-t-butyl-dithiomethyl-dCTP-SS-BodipyFL (3′-O-DTM-dCTP-SS-BodipyFL)(10). Compound 9 (1 mg) was dissolved in DMF (200u1), and DSC (0.4 mg)and DIPEA (0.6 ul) were added. After shaking the reaction mixture for anhour, it was added into a 300 ul solution of 3′-O—SS-dCTP-PA-NH₂(compound 9 in prior scheme) in 0.1 M Na₂CO3/NaHCO₃ buffer (pH 8.8). Themixture was shaken for 6 h, and 0.4 ml 0.1 M TEAC buffer (pH.8) wasadded. The resulting solution was subjected to DEAE ion exchangepurification. The column was eluted using TEAC buffer (pH.8) gradientfrom 0.1M to 0.8M. The product containing fraction was collected andconcentrated. HPLC was used to purify the final product (C-18 reversephase column, elution gradient: B from 0% in A to 50% in 40 min, A:HFIP-TEA buffer, B: Methanol). TOF-MALDI MS found (M+1): 1370; Cal:1365.

The synthesis of other three reversible terminators3′-O—SS-tert-butyl-dUTP—S—S-R6G (3′-O-DTM-dUTP-SS-R6G,3′-O—SS-tert-butyl-dATP—S—S-ROX (3′-O-DTA/I -dATP-SS-ROX), and3′-O—SS-tert-butyl-dGTP—S—S-Cy5 (3′-O-DTM-dGTP-SS-Cy5) followsessentially the same method as reported for3′-O—SS-/en-butyl-dCTP—S—S-BodipyFL (3′-O-DTM-dCTP-SS-BodipyFL).

Example 2 Cleavable Group Modified Nucleotide Analogues as ReversibleTerminators for DNA Sequencing by Synthesis

We designed and synthesized new dye labeled 3′-O-DTM dNTPs in which theDye-DTM moiety is more closely attached to the base through a shorterlinker (FIGS. 1A-1B, FIG. 2 ), so that after incorporation and cleavagea much smaller tail is left on the base, greatly facilitating upcomingenzymatic reactions. In addition, we have described the design andsynthesis of nucleotide analogues that are attached with small “anchor”moieties to the nucleobase via the DTM linker (FIG. 3B). Since attachingsmaller groups to the nucleobase will substantially decreaseinterference with recognition of these molecules as substrates bypolymerase, these NRTs are more efficiently incorporated into thegrowing DNA strand in SBS. After nucleotide incorporation, acorresponding labeled binding molecule tethered to a fluorescent dyewill orthogonally react with the anchor on the DNA extension product.Imaging of the fluorescent dye on this DNA extension product willidentify the incorporated nucleotide for sequence determination. Ageneral scheme to use these molecules for SBS is shown in FIG. 1B andFIGS. 4A-4D.

The anchor moieties include a variety of orthogonally reactive oraffinitive functionalities, such as biotin, azide, trans-cyclooctene(TCO) and phenyl boric acid (PBA) (FIG. 5 ), which will specifically andefficiently bind or react with streptavidin, dibenzocyclooctyne(DBCO),^(29,30) tetrazine (TZ)^(31,32) and salicylhydroxamic acid(SHA)³³ respectively (FIG. 6 , FIG. 7 ). The DNA polymerase will readilyincorporate these 3′-O-anchor-modified nucleotides to the growing DNAstrand to terminate DNA synthesis. Addition of the labeled bindingmolecules (such as different fluorophore-labeled streptavidin, DBCO, TZand SHA) to the corresponding primer extension product leads toorthogonal binding of the labeled binding molecules with thecorresponding “anchor” moiety on the primer extension product; afterwashing away the unbound labeled molecule, the detection of the uniquelabel attached to the 3′ end of the primer extension product determinesthe identity of the incorporated nucleotide. Labeling moieties cancontain multiple fluorophors enabling high sensitivity detection in SBS,potentially for single molecule SBS.

In addition to performing four-color SBS using the aforementionednucleotide analogues, these molecules also allow a wide spectrum of newDNA sequencing methods including one-color or two-color SBS at thesingle-molecule level or at an ensemble level. Instead of attaching asingle dye to the labeled binding molecules, multiple dyes can also beattached to the incorporated nucleotide through conjugation with labeledbinding molecules that carry multiple dyes (or dendrimers labeled withmultiple dyes) (FIGS. 9A-9C, FIG. 10 ), so that amplification offluorescent signals can be achieved to facilitate single-moleculedetection of the DNA extension product via SBS. The anchor labeled NRTsare especially advantageous in this regard. Small anchor tethered NRTstend to be incorporated by polymerases more efficiently, and thesubsequent labeling reactions using large substituents (such as dyelabeled dendrimers and FRET cassettes) (FIGS. 11A-11D, FIG. 12 ) willenable high sensitivity or even single molecule detection. Two-color SBScan be achieved by connecting a binding molecule to a FluorescenceResonance Energy Transfer (FRET) cassette formed by two differentfluorescent dyes, with distinct emissions, which generate four differentFRET signal signatures to identify the four DNA bases (A, C, G,T).^(34,35) If each labeled binding molecule is constructed byconjugation with a dye reporter using a uniquely cleavable linker forlabeling the DNA extension product, different cleavage methods can beused for the selective removal of the dye from the DNA extensionproduct; the detected signal changes will therefore determine theincorporated nucleotide at the single-molecule level, or at the ensemblelevel, to perform SBS. A well-established cleavable linker toolbox[azo,³⁶⁻³⁸ dimethylketal,^(39,40) Dde ((4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl)^(41,42) ally and nitrobenzyl^(3,4,8,9)] is available to develop the linkage between the labeledbinding molecules and the reporting dye. These linkers can be readilycleaved under specific conditions by mild treatment with sodiumdithionite (Na₂S₂O₄), weak acid, hydrazine (N₂H₄), Pd(0) andlight-irradiation, respectively.

In order to perfect the sequencing technology using the above mentioneddye or anchor labeled NRTs, we designed and synthesized 3′-O-DTM-dNTPsand their derivatives (cf., FIG. 33 , FIG. 34 ). These 3′-O-DTM-dNTPsare used in combination with the dye or anchor labeled 3′-O-DTM-dNTPs toachieve synchronization of DNA sequencing reactions^(8,10) and walkingstrategies⁴³ therefore greatly increase sequencing read length andaccuracy.

MALDI-TOF MS was used to analyze the DNA extension products resultingfrom the use of the abovementioned nucleotide analogues in polymerasereactions (FIGS. 29A-29H, FIG. 30B, and FIG. 31 ). We established thatthese 3′-O-DTM nucleotide analogues and their derivatives are efficientsubstrates for DNA polymerase to terminate the DNA synthesis. Theseresults also established that both the fluorophore (or anchor moiety)and the 3-O-DTM group are removable with high efficiency in a singlestep in aqueous solution. Furthermore, accurate 4-color sequencing datawere obtained using 3′-O—SS-dNTP-(SS)CleavableLinker-Dyes and3′-O-SS-dNTPs (FIGS. 24A-24B) on surface-immobilized DNA (FIG. 32 ).

A variety of new DNA sequencing methods based on the combinatorial useof 3′-O-DTM-dNTPs, 3′-O-dNTP-SS-Dye, 3′-O-dNTP-SS-Anchor and theirorthogonal reporter dye labeled binding molecule counterparts orcleavable reporter dye labeled binding counterparts are describedherein.

Descriptions of Methods for DNA SBS using 3′-O—DTM-dNTPs-SS-Label(Anchor). Combinatorial use of3′-O-SS(DINI)-dNTPs-SS-Dyc, 3′-O-SS(DTM)-dNTPs-SS-anchor and3′-O-SS(DTM)-dNTPs along with orthogonal binding molecules conjugatedwith fluorescent dyes (or conjugated with fluorescent dyes usingdifferent cleavable linkages) allows the construction of a wide spectrumof new methods for four-color, two-color and one-color DNA SBS at thesingle molecule level or the ensemble level.

One-Color DNA SBS (FIGS. 4A-4D). A one-color SBS scheme using3′-O-SS(DTM)-dNTPs-SS-Biotin and Cy5 labeled streptavidin is shown inFIGS. 4A-4D. The DNA polymerase incorporation reaction is conducted byusing one of the four 3′-O—SS-dNTPs-SS-Biotin (FIG. 3 ), followed by theaddition of the Cy5 labeled streptavidin and imaging to determine DNAsequences as described in STEP 1 through STEP 4 (as shown in FIG. 4.1and repeated in FIGS. 4.2, 4.3 and 4.4 (see FIGS. 4A-4D)). Each stepuses a different added nucleotide analogue and consists of three parts:(PART a) Add polymerase and one of the four 3′-O —SS-dNTPs-SS-Biotinfollowed by washing; if the added nucleotide is complementary to thenucleotide on the template immediately next to the 3′ end of the primer,then the added nucleotide will be incorporated into the primer toproduce a DNA extension product that has a Biotin at the 3′ end. (PARTb) Add Cy5 labeled streptavidin, which will bind to the biotin at the 3′end of the DNA extension product. (PART c) After washing away theunbound Cy5 labeled streptavidin, perform imaging to detect the Cy5signal for the identification of the incorporated nucleotide. Note thatin this single dye SBS approach, nucleotides are added one at a time.Only one of the four 3′-O—SS-dNTP-SS-Biotins will be incorporated andsubsequently labeled by Cy5-Streptavidin. Following STEP 4, addition ofTHP to the DNA extension products will cleave the disulfide bond andregenerate a free 3′—OH group on the 3′ end of the DNA extensionproducts. Simultaneously, the dye or anchor attached on the base of thenucleotide will be removed. Sequentially repeat the process, consistingof STEP 1 through STEP 4, followed by THP cleavage, for continuingsequence determination. Multiple Cy5 dyes can be attached tostreptavidin, enabling high sensitivity detection, such as singlemolecule SBS.

Four-Color DNA SBS (FIG. 8 ). SBS is performed using3′-O-SS(DTM)-dNTPs-SS-“anchor” (3′-O-t-Butyldithiomethyl (SS)-dATP-SS-TCO, 3′-O-t-Butyldith i ° methyl (SS)-dCTP-SS-PBA,3′-O-t-Butyldithiomethyl(SS)-dGTP-SS-Biotin,3′-O-t-Butyldithiomethyl(SS)-dUTP-SS-N3) (FIG. 5 ) and fourcorrespondingly matched dye labeled binding molecules (Rox-LabeledTetrazine, Alexa488-Labeled SHA, Cy5-Labeled Streptavidin, andR6G-Labeled Dibenzocyclooctyne) (FIG. 6 ). Addition of the DNApolymerase and the four 3′-O-SS(DTM)-dNTPs-SS-“anchor” (3′-O—SS-dATP-SS-TCO, 3′-O —SS-dCTP-SS-PBA, 3′-O—SS-dGTP-SS-Biotin and 3′-O—SS-dUTP-SS-N3) to the immobilized primed DNA template enables theincorporation of the complementary nucleotide analogue to the growingDNA strand to terminate DNA synthesis. After washing away theunincorporated nucleotide analogues, add the dye labeled bindingmolecules are added; these will specifically connect with each of thefour unique “anchor” moieties at the 3′-end of each DNA extensionproduct to enable the labeling of each DNA product terminated with eachof the four nucleotide analogues (A, C, G, T) with the four distinctfluorescent dyes. Detection of the unique flourescence signal from eachof the flourescent dyes on the DNA products allows for theidentification of the incorporated nucleotide. Next, treatment of theDNA products with THP cleaves the SS linker, leading to the removal ofthe fluorescent dye and the regeneration of a free 3′—OH group on theDNA extension product, which is ready for the next cycle of the DNAsequencing reaction (as shown in the subsequent steps of FIG. 8 ).

FIG. 7 shows the formation of the conjugates or complexes between DNAproducts produced by incorporating the “anchor” labeled nucleotides(3′-O-t-Butyldithiomethyl-dATP-SS-TCO,3′-O-t-Butyldithiomethyl-dCTP-SS-PBA,3′-O-t-Butyldithiomethyl-dGTP-SS-Biotin,3′-O-t-Butyldithiomethyl-dUTP-SS-N₃) with four correspondingly matchedlabeled binding molecules (Rox-Labeled Tetrazine, Alexa488-Labeled SHA,Cy5-Labeled Streptavidin, and R6G-Labeled Dibenzocyclooctyne).

Labeling molecules consisting of multiple dyes such as fluorescentdendrimers (FIGS. 9A-9C and FIG. 10 ), and fluorescent energy transfercassettes (FIGS. 11A-11D and 12 ) can be used to greatly increase thesensitivity of these SBS approaches as well as simplify the opticalset-up.

Two-Color DNA SBS (FIGS. 14A-14B). A scheme using3′-O-SS(DTM)-dNTPs-SS-Dye (3′-O—SS-dATP-SS-Rox and3′-O—SS-dCTP-SS-Alexa488), 3′-O-SS(DTM)-dNTPs-SS-Anchor(3′-O—SS-dUTP-SS-N3 and 3′-O—SS-dGTP-SS-TCO) and their corresponding dyelabeled binding molecules (Rox-Tetrazine & BodipyFL-Dibenzocyclooctyne)(FIG. 13 ) to perform 2-color DNA SBS is shown in FIG. 14 . Addition ofthe DNA polymerase and the four nucleotide analogues(3′-O—SS-dATP-SS-Rox, 3′-O—SS-dCTP-SS-Alexa488, 3′-O—SS-dUTP —SS-N₃ and3-O—SS-dGTP-SS-TCO) to the immobilized primed DNA template enables theincorporation of the complementary nucleotide analogue to the growingDNA strand to terminate DNA synthesis (STEP 1). After washing away theunincorporated nucleotide analogues, the fluorescent signal from Rox andAlexa488 is detected to identify the incorporated nucleotide as A(labeled with Rox) or C (labled with Alexa488). The dye labeled bindingmolecules (Rox-Tetrazinc & Alexa488-Dibenzocyclooctyne) arc then addedto the DNA extension products (STEP 2), which will specifically connectwith the two unique “anchor” moieties (TCO and N3) on the base at the3′-end of each DNA extension product, to enable the labeling of each DNAproduct terminated with each of the two nucleotide analogues (G and T)with two distinct fluorescent dyes (labeled with Rox for G and labeledwith Alexa488 for T). Detection of the unique, newly producedfluorescence signal from Rox or Alexa488 on the DNA extension products(in addition to the absence of signal from STEP 1), allows for theidentification of the newly-incorporated nucleotides as G or Trespectively. Next, treatment of the DNA products with THP cleaves theSS linker, leading to the removal of the fluorescent dye and theregeneration of a free 3′—OH group on the DNA extension product (STEP3), which is ready for the next cycle of the DNA sequencing reaction (asshown in the subsequent steps of FIGS. 14A-14B).

Use of 3′-O-CleavableGroup-dNTPs-CleavableLinker-Label,3′-O-CleavableGroup-dNTPs-CleavableLinker-Anchor and3′-O-CleavableGroup-dNTPs (FIGS. 16A-16B) combined with labeled bindingmolecules (FIGS. 15A-15B) that are conjugated with fluorescent dyes viadifferent cleavable linkers allows the construction of one-color SBS atthe single molecule or the ensemble molecule levels. After incorporatingthe 3′-O-DTM-dNTPs-SS-“anchor” and the 3′-O-DTM-dNTP, treatment withorthogonal labeled binding molecules conjugated with fluorescent dyes(ATTO647N, Cy5, Rox, etc.) via different cleavable linkages (Azo, Dde,Nitrobenzyl, Dimethylketal, etc.) (FIGS. 15A-15B) results in thelabeling of all incorporated nucleotides with an anchor on the base atthe 3′-end of the DNA extension products due to the specificanchor-binding molecule reaction. Sequential and specific cleavage,followed by imaging, are carried out to remove the dye from the 3′-endof the DNA extension products, allowing signal changes to be accuratelydetected. Each cleavage method only cleaves one type of linker which isuniquely attached to one of the labeled binding molecules, thereforeeach cleavage method can be used to encode one of the DNA bases on theircorreponding anchor moiety for that particular nucleotide analogue. Ingeneral, only three of the four DNA bases (A, C, G, T) are required tohave a label for selective detection. Once the first three of thesebases are labeled, the fouth one does not require a label to bedifferentiated from the other three for sequence determination, asexemplified in the following schemes.

Synthetic Method for Base-Linked Dithiomethyl Linker:

The structure of the cleavable dithiomethyl linker attached to the basemoiety is as follows:

The synthesis comprises:

reacting 3-trimethylsilanyl-prop-2-yn-1-ol with DMSO, acetic acid andacetic anhydride to provideTtrimethyl(3-((methylthio)methoxy)prop-1-yn-1-yl)silane;

contacting trimethyl(3-((methylthio)methoxy)prop-1-yn-1-yl)silane withsulfuryl chloride/cyclohexene followed by reaction with potassiumthiotosylate to produceS-(((3-(trimethylsilyl)prop-2-yn-1-yl)oxy)methyl)4-methylbenzenesulfonothioate;

contacting S-4(3-(trimethylsilypprop-2-yn-1-yl)oxy)methyl)4-methylbenzenesulfonothioate with2,2,2-trifluoro-N-(2-mercapto-2-methylpropyl) acetamide in the presenceof triethylamine, followed by;

contacting with tetrabutylammonium fluoride to form2,2,2-trifluoro-N-(2-methyl-2-(((prop-2-yn-1-yloxy)methyl)disulfanyl)propyl)acetamide

The method also provides:

reacting 5′-O-tBDMS -3′-O-polvmerase compatible cleavable blockinggroup-5(7)-Iodo-nucleoside with2,2,2-trifluoro-N-(2-methyl-2-(((prop-2-yn-1-yloxy)methyl)disulfanyl)propyl)acetamidein the presence of Pd(0), CuI, triethylamine which results in theformation of 5′-O-tBDMS-3′-O-polymerase compatible cleavable blockinggroup-2′-deoxynucleoside-5(7)-2,2,2-trifluoro-5-yDethynyl)oxy)methyl)disulfanyl)-2-methylpropypacetamide;

reacting the above product from step e) with tetrabutylammonium fluorideto remove 5′-O-t-BDMS group permitting the formation of 3′-O-polymerasecompatible cleavable blockinggroup-2′-deoxynucleoside-5(7)-2,2,2-trifluoro-5-yl)ethynyl)oxy)methyl)disulfanyl)-2-methylpropypacetamide;contacting the 3′-O-polymerase compatible cleavable blockinggroup-2′-deoxynucleoside-5(7)-2,2,2-trifluoro-5-yl)ethynyl)oxy)methyl)disulfanyl)-2-methylpropyl)acetamide produced in step f) with2-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one, tetrabutylammoniumpyrophosphate, tributylamine, I₂, pyridine, and NH₄OH under conditionpermitting the formation of 3′-O-polymerase compatible cleavableblockinggroup-5(7)-(34(1-amino-2-methylpropan-2-yl)disulfanyemethoxy)prop-1-yn-1-yl)-2′-deoxynucleoside-5′-triphosphate

One-Color DNA SBS Using Selective Linker Cleavage to Remove the Dye(FIGS. 17A-17B). 1. In the presence of DNA polymerase, three anchormodified nucleotides [3′—SS(DTM)-dATP-SS-N₃, 3′—SS(DTM)-dUTP-SS-TCO,3′-SS(DTM)-dCTP-SS-Biotin] and 3′-t-Butyl-SS(DTM)-dGTP, as shown in FIG.16 are added to the primed DNA templates to allow incorporation into theprimer. 2. The fluorescent label (ATTO647N, for example) is attached byadding DBCO-Azo-(—N═N-Linker)-ATTO647N, Tetrazine-Dde(Linker)-ATTO647N,and Streptavidin-ATTO647N (shown in FIGS. 16A-16B) to the DNA extensionproducts that contain the incorporated anchor modified nucleotideanalogues, which leads to the labeling of all the incorporatednucleotides (except G) at their base due to specific anchor-bindingmolecule interaction. 3. After washing, the first round of imaging isperformed, and the DNA products terminated with A, C and T all displaythe same color, while the DNA products that do not emit a signal areterminated by a G. 4. The first cleavage (I) is conducted by treatmentwith sodium dithionite (Na₂S₂O₄), which only cleaves the azo linkage toremove the fluorescent dye from the DNA products terminated with the Anucleotide. The second round of imaging is performed. If the fluorescentsignal disappears after cleavage I, the DNA products are determined ashaving incorporated an A nucleotide. 5. The second cleavage (II) isconducted by treatment with hydrazine (N₂H₄), which will cleave the Ddelinkage to remove the fluorescent dye from the DNA products terminatedwith the T nucleotide. The third round of imaging is performed. If thefluorescent signal disappears after cleavage II, the DNA products aredetermined as having incorporated a T nucleotide. The DNA products withunchanged fluorescent signals are identified by inference as beingterminated by a C nucleotide. 6. The third cleavage (III) is conductedwith THP to cleave the disulfide bond and remove the dye on C, so thechange of the signal after the THP treatment confirms the DNA productsas being terminated by a C nucleotide. Meanwhile, the THP treatment willalso cleave the DTM (SS) bond to regenerate free 3′—OH on all the DNAextension products, which are ready for subsequent cycles ofsingle-color DNA SBS. 7. Steps 1 to 6 are repeated to continuesubsequent cycles of single-color DNA SBS.

One-Color DNA SBS Using a Reduced Number of Selective Cleavage Reactionsto Remove the Dye (FIG. 19 )

In the presence of DNA polymerase, two nucleotides with an anchor on thebase [(3′-O-SS(DTM)-dUTP-SS-N₃, 3′-O-SS(DTM)-dCTP-SS-Biotin)],3′-O-SS(DTM)-dATP-SS-Rox and 3′-O-t-Butyl-SS(DTM)-dGTP, shown in FIG. 18are added to the primed DNA templates to allow incorporation into theprimer.

After washing, the first round of imaging is performed, and the DNAproducts terminated with an A nucleotide analogue display the Rox signaland therefore are determined as having incorporated an A nucleotide,while the other DNA products terminated at G, C, T will not display anyfluorescent signals.

The fluorescent label (Rox, for example) is attached to the DNA byadding DBCO-Azo-(—N═N-Linker)-Rox and Streptavidin-Rox (as shown in FIG.18 ) to the DNA extension products that contain the incorporated anchormodified nucleotide analogues, which leads to the labeling of all theincorporated nucleotides (except G) on the base at their 3′-end due tospecific anchor-binding molecule interaction.

After washing, the second round of imaging is performed, and the DNAproducts are terminated with A, C and T all display the same Rox signal,while the DNA products that do not emit a signal are terminated by a Gnucleotide.

The first cleavage (I) is conducted by treatment with sodium dithionite(Na₂S₂O₄), which only cleaves the azo linkage to remove the fluorescentdye Rox from the DNA products terminated with the T nucleotide. Thesecond round of imaging is performed. If the Rox fluorescent signaldisappears after cleavage I, the DNA products are determined as havingincorporated a T nucleotide.

The second cleavage (11) is conducted with THP to cleave the disulfidebond and remove the dye from the DNA extension products terminated withnucleotides A and C, so the change of the signal after the THP treatmentdetermines the DNA products as being terminated by a C nucleotide,because DNA products terminated by an A have already being determined inthe first round of imaging described above. Meanwhile, the THP treatmentwill also cleave the DTM (SS) bond to regenerate free 3′—OH on all theDNA extension products, which are ready for subsequent cycles ofsingle-color DNA SBS. Steps 1 to 6 are repeated to continue subsequentcycles of single-color DNA SBS.

One-Color DNA SBS with All Labeled Nucleotides Using Selective LinkerCleavage to Remove the Dye (FIGS. 21A-21F). 1. In the presence of DNApolymerase, three anchor modified nucleotides [3′-O-SS(DTM)-dGTP-SS-N₃,3′-O-SS(DTM)-dCTP-SS-Biotin, 3′-O-SS(DTM)-dUTP-SS-TCO)] and3′-O-SS(DTM)-dATP-SS-Rox, as shown in FIGS. 20A-20B are added to theprimed DNA templates to allow incorporation into the primer. 2. Afterwashing, the first round of imaging is performed, and the DNA productsterminated with an A nucleotide analogue display the Rox signal andtherefore are determined as having incorporated an A, while the otherDNA products terminated at G, C, T will not display any fluorescentsignals. 3. The fluorescent label (Rox, for example) is attached to theDNA by adding DBCO-Azo-(—N═N-Linker)-Rox, Tetrazine-Dde-Rox andStreptavidin-Rox (shown in FIGS. 20A-20B) to the DNA extension productsthat contain the incorporated anchor nucleotide analogues, which leadsto the labeling of all the incorporated nucleotides at their base due tospecific anchor-binding molecule interaction. 4. After washing, thesecond round of imaging is performed, and the DNA products terminatedwith A, G, T, C all display the same Rox signal. Subtraction of the Roxsignals from the DNA products determined in the first round of imagingas terminated at an A nucleotide reveals the DNA products terminated atG, T, C. 5. The first cleavage (I) is conducted by treatment with sodiumdithionite (Na₂S₂O₄), which only cleaves the azo linkage to remove thefluorescent dye Rox from the DNA products terminated with the Gnucleotide. The second round of imaging is performed. If the Roxfluorescent signal disappears after cleavage I, the DNA products aredetermined as having incorporated a G nucleotide. 6. The second cleavage(II) is conducted with hydrazine (N₂H₄), which will cleave the Ddelinkage to remove the fluorescent dye Rox from the DNA productsterminated with the T nucleotide. The third round of imaging isperformed. If the Rox fluorescent signal disappears after cleavage II,the DNA products are determined as having incorporated a T nucleotide.If the Rox fluorescent signal stays after cleavage II, the DNA productsare determined as having incorporated a C nucleotide. 7. The thirdcleavage (III) is conducted with THP to cleave the disulfide bond andremove the Rox dye from the DNA extension products terminated withnucleotides A and C, so the change of the signal after the THP treatmentverifies the DNA products as been terminated by a C nucleotide, becauseDNA products terminated by an A nucleotide have already being determinedin the first round of imaging described above. Meanwhile, the THPtreatment will also cleave the DTM (SS) bond to regenerate free 3′—OH onall the DNA extension products, which are ready for subsequent cycles ofsingle-color DNA SBS. Steps 1 to 7 are repeated to continue subsequentcycles of single-color DNA SBS.

One-Color DNA SBS Using Uniquely Cleavable Dye Labeled dNTPs (FIGS.23A-23D). 1. In the presence of DNA polymerase, the three3′-O-CleavableGroup-dNTPs- CleavableLinker-Label[3′-O-SS(DTM)-dATP-SS-Rox, 3′-O-SS(DTM)-dUTP-Allyl-Rox,3′-O-SS(DTM)-dCTP-Nitrobenzyl-Rox] and 3′-O-tButyl-SS-dGTP, as shown inFIG. 22 are added to the primed DNA templates to allow incorporationinto the primer. 2. After washing, the first round of imaging isperformed, and the DNA products terminated with C, T and A all displaythe same Rox signal, while the DNA products that do not emit a signalare terminated by a G. 3. The first cleavage (I) is conducted byphoto-irradiation at ˜350 nm to remove the fluorescent dye Rox from theDNA products terminated with the C nucleotide. The second round ofimaging is performed. If the Rox fluorescent signal disappears aftercleavage the DNA products are determined as having incorporated a Cnucleotide. 4. The second cleavage (II) is conducted with Pd (O), whichwill cleave the allyl linkage to remove the fluorescent dye Rox from theDNA products terminated with the T nucleotide. The third round ofimaging is performed. If the Rox fluorescent signal disappears aftercleavage 11, the DNA products are determined as having incorporated a Tnucleotide. If the Rox fluorescent signal stays after cleavage 11, theDNA products are determined as having incorporated an A nucleotide. 5.The third cleavage (III) is conducted with THP to cleave the disulfidebond and remove the Rox dye from the DNA extension prodcucts terminatedwith nucleotide A, so the change of the signal after the THP treatmentverifies the DNA products as being terminated by an A. Meanwhile, theTHP treatment will also cleave the DTM (SS) bond to regenerate free3′—OH on all the DNA extension products, which are ready for subsequentcycles of single-color DNA SBS. Steps 1 to 4 are repeated to continuesubsequent cycles of single-color DNA SBS.

All of the above example sequencing methods can be modified by includinga chasing step^(8,10) with unlabeled nucleotide reversible terminators,for instance by using the 3′-O-t-Butyl-SS-dNTPs described herein. Inthis procedure, 3′-O-t-Butyl-SS-dNTPs will be used to run polymeraseextension after each step of the polymerase extension reaction using3′-O-CleavableGroup-dNTPs-CleavableLinker-Label and3′-O-CleavableGroup-dNTPs-CleavableLinker-Anchor to ensure completeprimer extension at the 3′-end for ensemble SBS.

Four-Color DNA SBS with Chasing (FIGS. 25A-25F). A scheme using3′-O-SS(DTM)-dNTP-SS-Dye (3′-O-t-Butyldithiomethyl (SS)-dATP —SS-Rox,3′-O-t-Butyldithiomethyl (SS)-dCTP-SS-Alexa488,3′-O-t-Butyldithiomethyl(SS)-dGTP-SS-Cy5, 3′-O-t-Butyldithiomethyl(SS)-dUTP-SS-R6G) and four 3′-O-t-Butyldithiomethyl(SS)-dNTPs(3′-O-t-Butyldithiomethyl(SS)-dATP, 3′-O-t-Butyldithiomethyl (SS)-dCTP,3′-O-t-Butyldithiomethyl (SS)-dTTP and3′-O-t-Butyldithiomethyl(SS)-dGTP) (FIGS. 24A-24B) is shown in FIGS.25A-25F. Step 1, addition of DNA polymerase and the four3′-O-SS(DTM)-dNTP-SS-Dye (3′-O-t-Butyldithiomethyl(SS)-dATP-SS-Rox,3′-O-t-Butyldithiomethyl(SS)-dCTP-SS-Alexa488,3′-O-t-Butyldithiomethy1(SS)-dGTP-SS-Cy5 and3′-O-t-Butyldithiomethyl(SS)-dUTP-SS-R6G) to the immobilized primed DNAtemplate enables the incorporation of the complementary dye labelednucleotide analogue to the growing DNA strand. The growing DNA strand isterminated with each of the four nucleotide analogues (A, C, G, T) withthe four distinct fluorescent dyes. Step 2, addition of DNA polymeraseand four 3′4)4-Butyldithiomethyl(SS)-dNTPs(3′-O-t-Butyldithiomethyl(SS)-dATP, 3′-O-t-Butyldithiomethyl(SS)-dCTP,3′-O-t-Butyldithiomethyl(SS)-dTTP and 3′-O-t-Butyldithiomethyl(SS)-dGTP)to the immobilized primed DNA template enables the incorporation of thecomplementary 3′-O—SS-nucleotide analogue to the growing DNA strandwhich is not extended with one of the dye labeled3′-O-t-Butyldithiomethyl(SS)-dNTP in step 1, a process defined aschasing. The growing DNA strands are terminated with one of the fournucleotide analogues (A, C, G. T) with the four distinct fluorescentdyes or the one of the four chase nucleotide analogues (A, C, G, T)without dye. After washing away the unincorporated nucleotide analogues(Step 3), detection of the unique fluorescence signal from each of thefluorescent dyes on the DNA products allows for the identification ofthe incorporated nucleotide for sequence determination (Step 4). Next,in Step 5, treatment of the DNA products with THP cleaves the SS linker,leading to the removal of the fluorescent dye and the regeneration of afree 3′—OH group on the DNA extension products, which are ready for thenext cycle of the DNA sequencing reaction. The chasing NRTs without basemodifications are more efficiently incorporated into DNA than thebase-labeled NRTs, and after the removal of the 3′-blocking group, thereare no scars on the DNA extension products, both of which lead to higheraccuracy and long reads.

Four-Color DNA SBS without Chasing (FIGS. 26A-26F). A scheme using3′-O-SS(DTM)-dNTP-SS-Dye (3′-O-t-Butyldithiomethyl(SS)-dATP-SS-Rox,3′-O-t-Butyldithiomethyl(SS)-dCTP-SS-Alexa488,3′-O-t-Butyldithiomethyl(SS)-dGTP-SS-Cy5,3′-O-t-Buty—ldithiomethyl(SS)-dUTP-SS-R6G) (FIGS. 24A-24B) withoutchasing is shown in FIGS. 26A-26F. Step 1, addition of the DNApolymerase and the four 3′-O-SS(DTM)-dNTP-SS-Dye(3′-O-t-Butyldithiomethyl(SS)-dATP-SS-Rox,3′-O-t-Butyldithiomethyl(SS)-dCTP-SS-Alexa488,3′-04-Butyldithiomethyl(SS)-dGTP-SS-Cy5,3′-O-t-Butyldithiomethyl(SS)-dUTP-SS-R6G) to the immobilized primed DNAtemplate enables the incorporation of the complementary' nucleotideanalogue to the growing DNA strand. The growing DNA strand is terminatedwith each of the four nucleotide analogues (A, C, G, T) labeled with thefour distinct fluorescent dyes. After washing (Step 2) to removeunincorporated dye labeled nucleotide analogues, detection of the uniquefluorescent signal (Step 3) from each of the fluorescent dyes on the DNAproducts allows for the identification of the incorporated nucleotidefor sequence determination. Next, in Step 4, treatment of the DNAproducts with THP cleaves the SS linker, leading to the removal of thefluorescent dye and the regeneration of a free 3′—OH group on the DNAextension product, which is ready for the next cycle of the DNAsequencing reaction.

Four-Color DNA SBS with mixed labeled and unlabeled reversibleterminators (FIG. 27A-27B). SBS using 3′-O-5S(DTM)-dNTP-SS-Dye(3′-O-t-Butyldithiomethyl(SS)-dATP-SS-Rox, 3′-O-t-Butyldithiomethyl(SS)-dCTP —SS-Alexa488, 3′-O-t-Butyldithiomethyl(SS)-dGTP-SS-Cy5,3′-O-t-Butyldithiomethyl(SS)-dUTP-SS-R6G) and four3′-O-t-Butyldithiomethyl(SS)-dNTPs (3′-O-t-Butyldithiomethyl(SS)-dATP,3′-O-t-Butyldithiomethyl(SS)-dCTP, 3′-O-t-Butyldithiomethyl(SS)-dTTP and3′-O-t-Butyldithiomethyl(SS)-dGTP) (FIGS. 24A-24B). Step 1, addition ofthe DNA polymerase, the four 3′-O-SS(DTM)-dNTP-SS-Dye((3′-O-t-Butyldithiomethyl(SS)-dATP-SS-Rox,3′-O-t-Butyldithiomethyl(SS)-dCTP-SS-Alexa488,3′-O-t-Butyldithiomethyl(SS)-dGTP-SS-Cy5,3′-O-t-Butyldithiomcthyl(SS)-dUTP-SS-R6G) and four3′-O-t-Butyldithiomethyl(SS)-dNTPs (3 Butyldithiomethyl(SS)-dATP,3′-O-t-Butyldithiomethyl(SS)-dCTP, 3′-O-t-Butyldithiomethyl(SS)-dTTP and3′-O-t-Butyldithiomethyl(SS)-dGTP) to the immobilized primed DNAtemplate enables the incorporation of the complementary nucleotideanalogue to the growing DNA strand. The ratio of the labeled andunlabeled NRTs can range from below 1:9 to above 9:1. The growing DNAstrand is terminated with each of the four nucleotide analogues (A, C,G, T) with the four distinct fluorescent dyes or without dye labeling.After washing away (Step 2) the unincorporated nucleotide analogues,detection of the unique fluorescent signal (Step 4) from each of thefluorescent dyes on the DNA products allows for the identification ofthe incorporated nucleotide. Next, in Step 4, treatment of the DNAproducts with THP cleaves the SS linker, leading to the removal of thefluorescent dye and the regeneration of a free 3′—OH group on the DNAextension product, which is ready for the next cycle of the DNAsequencing reaction.

A combined walking and sequencing approach to obtain longer total readlength using a combination of labeled and unlabeled NRTs (FIGS.28A-28B). A walking approach is designed to obtain longer runs ofsequence than would be achievable with SBS alone. Among the issues thatmight prevent long reads are the presence of chemical “scars” on thebases of the incorporated nucleotides, which will eventually preventfurther extension, and the inevitable loss of synchronization inensemble SBS, even with chase-style “fill-in” reactions. Briefly, asschematized in FIGS. 28A-28B, sequencing by synthesis reactions arecarried out to the desired length (30-100 or more nucleotides), forinstance by using any of the above described strategies (1-color,2-color or 4-color) and nucleotide analogues, or any other SBSsequencing approaches. Subsequently, the extended primer is strippedfrom the template strand and the original primer is re-annealed to it.Next, three natural nucleotides and one reversible terminator (dATP,dCTP, dTTP and 3′-O-t-butyl-SS-dGTP as an example), all without anyfluorescent labels, are used to extend the primer to the position of thenext base in the template that is complementary to the reversibleterminator (a C in this example). After cleavage of the blocking group(with THP in this example) to regenerate the 3′ OH group, an identicalextension reaction is performed to reach the subsequent C in thetemplate, and the resulting 3′ blocking group is again removed. Repeatedrounds of walking and cleavage will be used to approach the positionwhere the first sequencing round ended (this is estimated based on thepercent G's in the genome being sequenced). A second round of SBS withfluorescent NRTs is then carried out to obtain a further sequencestretch. If desired, the denaturation step is then repeated to strip offthe extended primer and replace it again with the original primer. A newround of walking is carried out to reach the end of the second sequencestretch. This process can be repeated several times, resulting in asequence twice, three times or higher-fold than that obtained in oneround of sequencing. The blocking nucleotide for the walking steps canbe replaced with an alternative one (e.g., 3′-O-t-butyl-SS-dATP alongwith dCTP, dGTP and dTTP, etc.). Alternative variants could include theuse of two, three or four 3′-blocked nucleotide with two, one or zeronatural nucleotides. Importantly, the walking steps will result inextension of the primer with only natural nucleotides, so very longwalks to reach the appropriate vicinity for the third or fourth round ofsequencing should not be problematic. An actual series of walking stepswith one 3′-O-DTM blocked nucleotide and three natural nucleotides isshown in FIG. 31 .

Polymerase extension using reversible terminators 3′—SS-dATP-SS-Rox,3′—SS-dCTP-SS-Alexa488, 3′—SS-dGTP-SS-Cy5, and 3′—SS-dUTP-SS-R6G andcharacterization by MALDI-TOF mass spectrometry (FIGS. 29A-29H).Extension reactions were carried out using 200 pmol of reversibleterminator, 2 units of Therminator IX DNA Polymerase (NEB), 20 pmol ofDNA primer (M.W. 6084), 100 pmol of DNA template in 20 ul buffercontaining 20 mM Tris-HCl, 10 mM (NH₄)₂SO₄, 10 mM KCl, 2 mM MgSO₄, 0.1%Triton X-100, pH 8.8@25° C., and 2 mM MnCl₂. The reactions wereconducted in an ABI GeneAmp PCR System 9700 with initial incubation at65° C. for 30 second, followed by 38 cycles of 65° C./30 sec, 45° C./30sec, 65° C./30 sec. The reaction mixtures were desalted using OligoClean & Concentrator™ (ZYMO Research) and analyzed by MALDI-TOF MS (ABIVoyager DE). The cleavage reactions were carried out using THP at afinal concentration of 5 mM, incubating at 65° C. for 10 minutes, thenthe reaction mixtures were desalted using Oligo Clean & Concentrator™(ZYMO Research) and analyzed by MALDI-TOF MS. The results of eachindividual extension and cleavage are shown in FIGS. 29A-29H, indicatingthat all these nucleotide analogues are efficient substrates for thepolymerase, with complete extension and cleavage.

Each of the four different primers indicated below were designed toallow extension by a different nucleotide (A, C, T or G from top tobottom). Exong_template (SEQ ID NO:4) was used for A, C and Textensions; Exon? template (SEQ ID NO:5) was used for G extension. Thepresence of two identical complementary bases in a row at the extensionsite on the template was a built-in control to account for anyincomplete termination.

Exon8_template: (SEQ ID NO: 4)5′-AAGGAGACACGCGGCCAGAGAGGGTCCTGTCCGTGTTTGTGCGTGGAGTTCGACAAGGCAGGGTCATCTAATGGTGATGAGTCCTATCCTTTTCTCTTC GTTCTCCGT- 3′.Exon7_template: (SEQ ID NO: 5)5′-TACCCGGAGGCCAAGTACGGCGGGTACGTCCTTGACAATGTGTACATCAACATCACCTACCACCATGTCAGTCTCGGTTGGATCCTCTATTGTGTCCGG G-3′.

The following 3 primers are used for extension with Exon_8 template: i)5′-TAGATGACCCTGCCTTGTCG-3′(SEQ ID NO:6); ii) 5′-TCTCTGGCCGCGTGTCT-3′(SEQID NO:3); iii) 5′-GATAGGACTCATCACCA-3′(SEQ ID NO:7). The followingprimer is used for extension with Exon 7 template:5′-GTTGATGTACACATTGTCAA-3′(SEQ ID NO:8).

Continuous Polymerase Extension Using 3′-O-t-Butyl-SS-dNTPs(3′-O-DTM-dNTPs) and Characterization by MALDI-TOF Mass Spectrometry(FIGS. 30A-30B). To verify that the 3′-O-DTM-dNTPs are incorporatedaccurately in a base-specific manner in the polymerase reaction, fourconsecutive DNA extension and cleavage reactions were carried out insolution with 3′-O-DTM-dNTPs as substrates. This allowed the isolationof the DNA product at each step for detailed molecular structurecharacterization.

We performed a complete consecutive 4-step SBS reaction that involvedincorporation of each complementary 3′-O-DTM-dNTP, followed by MALDI-TOFMS analysis for sequence determination, and cleavage of the 3′-O-DTMblocking group from the DNA extension product to yield a free 3′—OHgroup for incorporating the next nucleotide analogue. A template-primercombination was designed in which the next four nucleotides to be addedwere A, C, G and T. As shown in FIG. 30 . the SBS reaction was initiatedwith the 13-mer primer annealed to a DNA template. When the firstcomplementary nucleotide, 3′-O-tButyl-SS-dATP (3′-O-DTM-dATP), was usedin the polymerase reaction, it was incorporated into the primer to forma DNA extension product with a molecular weight of 4404 Daltons (Da) asconfirmed by MALDI-TOF MS with the appearance of a single peak (FIG. 30B(a) Top left). These results indicated that the 3′-O-DTM-dATP wasquantitatively incorporated into the 13-mer DNA primer. After THPtreatment to remove the DTM group from the DNA product and HPLCpurification, the cleavage was confirmed by the presence of a single MSpeak at 4272 Da, corresponding to the DNA product with the 3′-O-DTMgroup removed (FIG. 30B (b)Top right). The newly formed DNA extensionproduct with a free 3′—OH group was then used in a second polymerasereaction to incorporate a 3′-O-1-Butyl-SS-dCTP (3′-O-DTM-dCTP) whichgave a single MS peak at 4697 Da (FIG. 30B (c)), indicatingincorporation of a 3′-O-DTM-dCTP into the growing DNA strand in thiscycle. After THP treatment, a single MS peak of the cleaved DNA productappeared at 4563 Da (FIG. 30B (d)), which demonstrated the completeremoval of the DTM group from the DNA extension product.

The third incorporation was with 3′-O -Butyl —SS-dGTP (3′-O-DTM-dGTP);accurate masses of the corresponding DNA products were obtained byMALDI-TOF MS for the third nucleotide incorporation (5024 Da, FIG. 30B(e), and cleavage reaction (4888 Da, FIG. 30B (f)). Finally,3′-O-tButyl-SS-dTTP (3′-O-DTM-dTTP) incorporation in the fourth cycleand a final removal of the DTM group by THP was verified, as appropriatemasses for the corresponding DNA products were obtained by MALDI-TOF MSfor the fourth nucleotide incorporation (5328 Da, FIG. 30B (g)) andcleavage reaction (5199 Da, FIG. 30B (h)). These results demonstratethat all four 3′-O-DTM-dNTPs are efficiently incorporatedbase-specifically as reversible terminators into the. growing DNA strandin a continuous pOlyMeraSe reaction, and that the 3′—OH capping, groupon the DNA extension products is quantitatively cleaved by THP.

Experiment demonstrating walking in solution using three natural dNTPs(dATP, dCTP and dTTP) and one 3′-O-t-Butyl-SS-dNTP (3′-O-DTM-dGTP) (FIG.31 ). We carried out a series of 3 walking steps using dATP, dCTP, dTTPand 3′-O-t-butyl-SS-dGTP. The results are presented in FIG. 31 . WT49G(5′-CAGCTTAAGCAATGGTACATGCCTTGACAATGTGTACATCAACATCACC-3′) (SEQ IDNO:10)was designed as template for a 1^(st) walk extension of 4 bases onthe primer (13mer, 5′-CACATTGTCAAGG-3′) (SEQ ID NO:2), 8 base extensionin the 2^(nd) walk and 6 base extension in the 3^(rd) walk; in eachcase, the reaction will stop at the first corresponding C on thetemplate (shown in red from right to left in the template). The WT49Gtemplate and 13mer primer were designed for efficient characterizationof walking by MALDI-TOF mass spectrometry.

The reaction (50 ul) was carried out using 1pmol of reversibleterminator, 1 umol of dATP, dCTP and dTTP, 500 pmol of primer (MM.3939), 5 units of Therminator IX DNA Polymerase (NEB), 300 pmol of WT49Gin a 5 μl buffer containing 20 mM Tris-HCl, 10 mM (NH₄)₂SO₄, 10 mM KCl,2 mM MgSO₄, 0.1% Triton X-100, pH 8.8 (4), 25° C., and 100 pmol MnCl₂.The reactions were conducted in an ABI GeneAmp PCR System 9700 withinitial incubation at 65° C. for 30 seconds, followed by 38 cycles of65° C./30sec, 45° C./30 sec, 65° C./30 sec. the reaction mixtures weredesalted using Oligo Clean & Concentrator™ (ZYMO Research) and analyzedby MALDI-TOF MS (ABT Voyager DE). The cleavage reaction was carried outusing THP at a final concentration of 5 mM incubated at 65° C. for 5minutes, then the reaction mixtures were desalted using oligo Clean &Concentrator™ (ZYMO Research) and analyzed by MALDI-TOF MS. The resultsof each individual extension and cleavage are shown in FIG. 31 .

After the first walk, the primer was extended to the point of the next Cin the template (rightmost C highlighted in red in the template strand).The size of the extension product was 5330 Daltons (5328 Da expected) asshown in the top left MALDI-TOF MS trace. After cleavage with THP, the5198 Da product shown at the top right was observed (5194 Da expected).A second walk was performed using this extended and cleaved primer,again using Therminator IX DNA polymerase, dATP, dCTP, dTTP and3′-O-t-butyl-dGTP, to obtain the product shown in the middle left trace(7771 Da observed, 7775 Da expected to reach the middle C). Aftercleavage, a product of 7643 Da was obtained (expected 7641 Da). Finallya third walk and cleavage using the previously extended and cleavedprimer were performed, giving products of 9625 Da (9628 Da expected toextend to the leftmost red highlighted C) and 9513 Da (9493 Daexpected), respectively. The amount of nucleotides was adjusted in eachwalk according to extension length (2 μmol in 2^(nd) walk, 1.5 μmol in3^(rd) walk). This demonstrates the ability to use a 3′4)-t-butylnucleotide as a terminator for walking reactions. These can beincorporated into a combined sequencing and walking scheme such as theone depicted in FIGS. 28A-28B.

Experiment demonstrating four-color SBS on surface-immobilized DNA (FIG.32 ). The 5′-amino modified self-priming template DNA(5′-CACTCACATATGTTTTTTAGCTTTTTTAATTTCTTAATGATGTTGTTGCATGCTGACCTCAGCTGCACGTAAGTGCAGCTGAGGTCAG-3′) (SEQ ID NO:1) was dissolved in 50 mMsodium phosphate buffer, pH 9.0, at a concentration of 30 μM and spottedon NHS ester-activated CodeLink slides (Surmodics Inc., MN) using aSpotArray 72 microarray-printing robot (PerkinElmer, MA). Afterspotting, the slides were incubated overnight at 37° C. in a humidchamber containing a solution of saturated sodium chloride to immobilizethe DNA. Upon immobilization, unreacted NHS ester groups were quenchedby incubating the slides in a solution of 50 mM 3-amino-1-propanol in100 mM tris-HCl buffer, pH9.0 for 2 hours at ambient temperature.Finally, the slides were briefly rinsed in water, air-dried undercompressed air and stored desiccated in a dark container until furtheruse.

The slide was then covered with a silicone isolator and 8 μl ofextension mixture containing four reversible terminators(3′—SS-dARP-SS-Rox, 3′-SS-dCTP —SS-Alexa488, 3 ′—SS-dGTP-SS-Cy5, and3′—SS-dUTP-SS-R6G) (FIGS. 24A-24B), 5 units of Therminator IX DNAPolymerase (NEB), 1× Thermo Pol Reaction Buffer (NEB). 2 mM MnCl₂ wasadded to the area coated with self-priming DNA template. The slide wasincubated at 65° C. for 15 minutes then washed with lx Thermo PolReaction Buffer (NEB), 2 mM MnCl₂ twice at room temperature. Eightmicroliters of a chase mixture containing 1 μM each of 3′—SS-dNTPs(FIGS. 24A-24B), 5 units of Therminator TX DNA Polymerase (NEB), lxThermo Pol Reaction Buffer (NEB), 2 mM MnCl₂ was then added andincubation carried out at 65° C. for 15 minutes. The silicone isolatorwas then removed and the slide was washed with SPSS buffer containing 2%Tween 20 at 37° C. for 30 minutes then rinsed with distilled water.Imaging was carried out using a ScanArray Express Microarray Scanner(Perkin Elmer). Four channel scanning using excitation at 488 nm, 543nm, 594 nm, and 633 nm was performed, and the fluorescence intensity wasrecorded.

The slide was again covered with a silicone isolator and 8 μl of THP at5 mM in 1× PBS was added and incubated at 65° C. for 15 minutes toremove the fluorescent dye and reestablish the hydroxyl group at the 3′end. The silicone isolator was removed again and the slide was washedwith SPSS buffer containing 2% Tween 20 and re-scanned to confirmsuccessful removal of the dye on the base along with the 3′ blockinggroup. The above procedure was repeated for each of the subsequentsequencing cycles.

Four-color sequencing data on a surface are shown in FIG. 32 . Theintensity of the emitted light at the following four wavelengths (488nm, 543 nm, 594 nm, and 633 nm) is indicated by the height of the bars(first: Alexa 488=C; second: R6G =T; third: Rox =A; fourth: Cy5 =G,respectively). From left to right, the sets of 4 bars are for the P^(t)extension reaction, the 1^(st) cleavage reaction, the 2^(nd) extensionreaction, the 2¹′^(d) cleavage reaction, the 3^(rd) extension reaction,the 3^(rd) cleavage reaction, the 4^(th) extension reaction, and the4^(th) cleavage reaction. The expected fluorescence emission wavelengthwas obtained for each cycle of nucleotide addition based on thenucleotide at that position in the template, demonstrating excellentfidelity with this combination of labeled, blocked nucleotides(3′—SS-dARP-SS-Rox, 3′—SS-dCTP-SS-Alexa488, 3′—SS-dGTP-SS-Cy5, and3′-SS-dUTP-SS-R6G) and Therminator IX polymerase. Furthermore, theemission was reduced to background after treatment with THP, indicatingcomplete cleavage of the dye from the base.

3′-O-methylthiomethyl-5′-O-iert-butyldimethylsilyl thymidine (2a): To astirring solution of the 5′-O-tent-butyldimethylsilyl thymidine (la,1.07 g, 3 mmol) in DMSO (10 mL) was added acetic acid (2.6 mL, 45 mmol)and acetic anhydride (8.6 mL, 90 mmol). The reaction mixture was stirredovernight at room temperature. Then the mixture was added slowly to asaturated solution of sodium bicarbonate under vigorous stirring andextracted with ethyl acetate (3×30 mL). The combined organic layers weredried over Na₂SO₄ and filtered. The filtrate was concentrated to drynessunder reduced pressure and the compound was purified by silica gelcolumn chromatography (ethyl acetate/hexane: 1:2) to give pure product2a (0.97 g, 74%). ¹H NMR (400 MHz, CDCl₃) δ: 8.16 (s, 1H), 7.48 (s, 1H),6.28 (m, 1H),4.62 (m, 2H), 4.46 (m, 1H), 4.10 (m, 1H), 3.78-3.90 (m,2H), 2.39 (m, 1H), 2.14 (s, 3H), 1.97 (m, 1H), 1.92 (s, 3H), 0.93 (s,9H),0.13 (s, 3H); HRMS (FAB⁺) calc'd for C₁₈H₃₃N₂O₅SSi [(M+H)+]:417.1879, found: 417.1890.

3′-O-tert-butyldithiomethyl-5′-O-tert-butyldimethylsilyl thymidine (3a):3′-O-methylthiomethyl-5′-O-tert-butyldimethylsilyl thymidine (2a, 420mg, 1 mmol) was dissolved in anhydrous dichloromethane (20 mL), followedby addition of triethylamine (0.18 mL, 1.31 mmol, 1.2 eq.) and molecularsieves (3 Å, 2 g). The mixture was cooled in an ice bath after stirringat room temperature for 30 min and then a solution of sulfuryl chloride(redistilled, 0.1 mL, 1.31 mmol, 1.2 eq.) in anhydrous dichloromethane(3 mL) was added dropwise over 2 minutes. The ice bath was removed andthe reaction mixture was stirred further for 30 min. Then potassiump-toluenethiosulfonate (375 mg, 1.65 mmol) in anhydrous DMF (2 mL) wasadded to the mixture. Stirring was continued at room temperature for anadditional hour followed by addition of tent-butyl mercaptan (1 mL). Thereaction mixture was stirred at room temperature for 30 min and quicklyfiltered through celite. The filter was washed with dichloromethane andthe organic fraction was concentrated to give crude product 3a.

3′-O-tert-butyldithiomethyl-thymidine (4a): Without isolation, the crudecompound 3a was dissolved in THF (10 mL) and a THF solution oftetrabutylammonium fluoride (1.0M, 1.04 mL 1.04 mmol) was added. Thereaction mixture was stirred at room temperature for 4 hours. Thereaction mixture was concentrated in vacuo, saturated NaHCO₃ solution(50 mL) was added and the mixture was extracted with dichloromethane(3×20 mL). The organic layer was dried over anhydrous Na₂SO₄, filtered,concentrated and the obtained crude mixture was purified by flash columnchromatography (dichloromethane/methanol: 20:1) to give3′-O-Cert-butyldithiomethyl-thymidine 4a (132 mg, 35% from compound 2a).¹H NMR (300 MHz, CDCl₃) δ: 7.41 (q, J=1.2 Hz, 1H), 6.15 (dd, J=7.4, 6.5Hz, 1H), 4.89-4.82 (in, 2H), 4.62-4.54 (m, 1H), 4.15 (q, J=3.0 Hz, 1H),3.97-3.86 (m, 2H), 2.42 (ddd, J=7.5, 4.8, 2.5 Hz, 2H), 1.95 (d, J=1.2Hz, 3H), 1.36 (s, 8H).

3′-O-tert-butyldithiomethyl-dTTP (5a):3′-O-Cert-butyldithiomethyl-thymidine (4a, 50 mg, 0.13 mmol),tetrabutylammonium pyrophosphate (197 mg, 0.36 mmol) and2-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one (44 mg, 0.22 mmol) weredried separately overnight under high vacuum at ambient temperature. Thetetrabutylammonium pyrophosphate was dissolved in dimethylformamide(DMF, 1 mL) under argon followed by addition of tributylamine (1 mL).This mixture was injected into the solution of2-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one in (DMF, 2 mL) underargon. After stirring for 1 h, the reaction mixture was added to thesolution of 3′-O-tert-butyldithiomethyl-thymidine and stirred furtherfor 1 hour at room temperature. Iodine solution (0.02 Miodine/pyridine/water) was then injected into the reaction mixture untila permanent brown color was observed. After 10 min, water (30 mL) wasadded and the reaction mixture was stirred at room temperature for anadditional 2 hours. The resulting solution was extracted with ethylacetate (2×30 mL). The aqueous layer was concentrated under vacuum andthe residue was diluted with 5 ml of water. The crude mixture was thenpurified with anion exchange chromatography on DEAE-Sephadex A-25 at 4°C. using a gradient of TEAB (pH 8.0; 0.1-1.0 M). The crude product wasfurther purified by reverse-phase HPLC to afford 5a, which wascharacterized by MALDI-TOF MS: calc'd for C₁₅H₂₇N₂O₁₄P₃S₂: 616.4, found:615.4.

N²-isobutyryl-3′-O-methylthiomethyl-5′-O-tert-butyldimethylsilyl-T-deoxyguanosine(G2): To a stirring solution ofN²-isobutyryl-5′-O-Cert-butyldimethylsilyl-2′-deoxyguanosine (G1, 1.31g, 3 mmol) in DMSO (10 mL) was added acetic acid (2.6 mL, 45 mmol) andacetic anhydride (8.6 mL, 90 mmol). The reaction mixture was stirred atroom temperature until the reaction was complete, which was monitored byTLC. Then the mixture was added slowly to a saturated solution of sodiumbicarbonate under vigorous stirring and extracted with ethyl acetate(3×30 mL). The combined organic layers were dried over Na₂SO₄ andfiltered. The filtrate was concentrated to dryness under reducedpressure and the compound was purified by silica gel columnchromatography (DCM/methanol: 20:1) to give pure product G2 (75%, 1.15g). ¹H NMR (400 MHz, CDCl₃) δ12.10 (d, J=2.9 Hz, 1H), 9.17 (d, J=3.0 Hz,1H), 8.03 (m, 1H), 6.18 (td, J=6.9, 2.9 Hz, 1H), 4.74-4.60 (m, 3H), 4.13(dq, J=6.8, 3.3 Hz, 1H), 3.84-3.75 (m, 2H), 2.78 (m, 1H), 2.54 (m, 2H),2.16 (s, 3H), 1.33-1.22 (m, 6H), 0.96-0.87 (m, 9H), 0.09 (dd, J=6.7, 3.8Hz, 6H).

N²-isobutyryl-3′-O-tert-butyldithiomethyl-5′-O-tert-butyldimethylsilyl-2′-deoxyguanosine(G3):N²-isobutyryl-3′-O-methylthiomethyl-5′-O-tert-butyldimethylsilyl-2′-deoxyguanosine(G2, 511 mg, 1.0 mmol) was dissolved in anhydrous dichloromethane (20mL), followed by addition of triethylamine (0.17 mL, 1.2 mmol) andmolecular sieves (3 A, 2 g). The mixture was cooled in an ice bath afterstirring at room temperature for 30 min and then a solution of sulfurylchloride (0.095 mL, 1.2 mmol) in anhydrous dichloromethane (3 mL) wasadded dropwise over 2 minutes. The ice bath was removed and the reactionmixture was stirred further for 30 min. Then potassium4-toluenethiosulfonate (341 mg, 1.5 mmol) in anhydrous DMF (2 mL) wasadded to the mixture. Stirring was continued at room temperature for anadditional hour followed by addition of tert-butyl mercaptan (1 mL). Thereaction mixture was stirred at room temperature for 30 min and quicklyfiltered through celite. The filter was washed with dichloromethane andthe organic fraction was concentrated to give crude product G3.

N²-isobutyryl-3′-O-tert-butyldithiomethyl-2′-deoxyguanosine (G4).Without isolation, the crude compound G3 was dissolved in THF (10 mL)and a THF solution of tetrabutylammonium fluoride (1.0M, 1.04 mL, 1.04mmol) was added. The reaction mixture was stirred at room temperaturefor 4 hours. The reaction mixture was concentrated in vacuo, saturatedNaHCO₃ solution (50 mL) was added and the mixture was extracted withdichloromethane (3×20 mL). The organic layer was dried over anhydrousNa₂SO₄, filtered, concentrated and the obtained crude mixture waspurified by flash column chromatography (dichloromethane/methanol: 20:1)to give N²-isobutyryl-3′-O-tert-butyldithiomethyl-2′-deoxyguanosine G4(155 mg, 33% from compound G2). ¹H NMR (400 MHz, CDCl₃) δ12.19 (s, 1H),9.44 (s, 1H), 7.97 (s, 1H), 6.17 (dd, ./=8.4, 5.9 Hz, 1H), 5.04 (s, 1H),4.92-4.80 (m, 2H), 4.76-4.64 (m, 1H), 4.26 (q, J=2.6 Hz, 1H), 3.98 (dd,J=12.2, 2.8 Hz, 1H), 3.80 (d, J=12.3 Hz, 1H), 2.91-2.73 (m, 2H), 2.49(in, 1H), 1.35 (s, 9H), 1.36-1.22 (in, 6H). ¹³C NMR (75 MHz, CDCl₃)δ179.60, 155.80, 148.10, 147.96, 139.11, 122.30, 86.29, 81.22, 78.96,63.21, 48.07, 38.18, 36.64, 30.29, 19.39, 19.34.

3′-O-tert-butyldithiomethyl-dGTP (G5).N²-isobutyryl-3′-O-tert-butyldithiomethyl-2′-deoxyguanosinc (G4, 50 mg,0.11 mmol), tetrabutylammonium pyrophosphate (180 mg, 0.33 mmol) and2-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one (44 mg, 0.22 mmol) weredried separately overnight under high vacuum at ambient temperature. Thetetrabutylammonium pyrophosphate was dissolved in dimethylformamide(DMF, 1 mL) under argon followed by addition of tributylamine (1 mL).This mixture was injected into the solution of2-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one in (DMF, 2 mL) underargon. After stirring for 1 h, the reaction mixture was added to thesolution of N²-isobutyryl-3′-O-tort-butyldithiomethyl-2′-deoxyguanosineand stirred further for 1 hour at room temperature. Iodine solution(0.02 M iodine/pyridine/water) was then injected into the reactionmixture until a permanent brown color was observed. After 10 min, water(30 mL) was added and the reaction mixture was stirred at roomtemperature for an additional 2 hours. The resulting solution wasextracted with ethyl acetate. The aqueous layer was concentrated invacuo to approximately 20 mL, then concentrated NH₄OH (20 ml) was addedand the mixture stirred overnight at room temperature. The resultingmixture was concentrated under vacuum and the residue was diluted with 5ml of water. The crude mixture was then purified with anion exchangechromatography on DEAE-Sephadex A-25 at 4° C. using a gradient of TEAB(pH 8.0; 0.1-1.0 M). The crude product was further purified byreverse-phase HPLC to afford G5. HRMS (ESI) calc'd for C₁₅H₂₅N₅O₁₃P₃S₂[(M−H)⁻]: 640.0103, found: 640.0148.

N⁶-Benzoyl-5′-O-tert-butyldimethylsilyl-3′-O-methylthiomethyl-2′-deoxyadenosine(A2): To a stirring solution of theN⁶-Benzoyl-5′-O-tert-butyldimethylsilyl-2′-deoxyadenosine (Al, 1.41 g, 3mmol) in DMSO (10 mL) was added acetic acid (3 mL) and acetic anhydride(9 mL). The reaction mixture was stirred at room temperature until thereaction was complete, which was monitored by

TLC. Then the mixture was added slowly to a solution of sodiumbicarbonate under vigorous stirring and extracted with ethyl acetate(3×30 mL). The combined organic layers were dried over Na₂SO₄ andfiltered. The filtrate was concentrated to dryness under reducedpressure and the residue of the desired compound was purified by silicagel column chromatography (dichloromethane/methanol: 30:1) to give pureproduct A2 (1.39 g, 88%). ¹H NMR (400 MHz, CDCl₃) δ9.12 (s, 1H), 8.81(s, 1H), 8.35 (s, 1H), 8.10-8.01 (m, 2H), 7.68(m, 1H), 7.49(m, 2H), 6.53(dd, J=7.5, 6.0 Hz, 1H), 4.78-4.65 (m, 3H), 4.24 (dt, J=4.3, 3.1 Hz,1H), 3.98-3.81 (m, 2H), 2.80-2.60 (m, 2H), 2.21 (s, 3H), 0.94 (s, 10H),0.13 (s, 6H): MS (APCI⁺) calc'd for C₂₆H₃₆N₄O₄SSi: 528.74, found: 529.4[M+H]⁺.

N⁶-B en zoyl -5′-O-tert-butyl dim ethyl silyl-3′-O-tert-butyldithiomethyl-2′-deoxyadenosine (A3):N⁶-Benzoyl-5′-O-tert-butyldimethylsilyl-3-O-methylthiomethyl-2′-deoxyadenosine (A2, 529 mg, 1.0 mmol) was dissolved in anhydrous dichloromethane(20 mL), followed by addition of triethylamine (0.17 mL, 1.2 mmol) andmolecular sieves (3 A, 2 g). The mixture was cooled in an ice bath afterstirring at room temperature for 30 min and then a solution of sulfurylchloride (0.095 mL, 1.2 mmol) in anhydrous dichloromethane (3 mL) wasadded dropwise over 2 minutes. The ice bath was removed and the reactionmixture was stirred further for 30 min. Then potassium4-toluenethiosulfonate (341 mg, 1.5 mmol) in anhydrous DMF (2 mL) wasadded to the mixture. Stirring was continued at room temperature for anadditional hour followed by addition of tert-butyl mercaptan (1 mL). Thereaction mixture was stirred at room temperature for 30 min and quicklyfiltered through celite. The filter was washed with dichloromethane andthe organic fraction was concentrated to give crude product A3.

N⁶-Benzoyl-3′-O-tert-butyldithiomethyl-2′-deoxyadenosine (A4): Withoutisolation, the crude compound A3 was dissolved in THF (10 mL) and a THFsolution of tetrabutylammonium fluoride (1.0M, 1.04 mL, 1.04 mmol) wasadded. The reaction mixture was stirred at room temperature for 4 hours.The reaction mixture was concentrated in vacuo, saturated NaHCO₃solution (50 mL) was added and the mixture was extracted withdichloromethane (3×20 mL). The organic layer was dried over anhydrousNa₂SO₄, filtered, concentrated and the obtained crude mixture waspurified by flash column chromatography (dichloromethane/methanol: 20:1)to give N⁶-Benzoyl-3′-O-tert-butyldithiomethyl-2′-deoxyadenosine A4 (128mg, 26% from compound A2). ¹H NMR (400 MHz, DMSO-d₆) δ11.18 (s, 1H),8.77 (s, 1H), 8.71 (s, 1H), 8.10-8.02 (in, 2H), 7.66 (t, J=7.6 Hz, 1H),7.56 (t, J=7.6 Hz 2H), 6.47 (dd, J=8.0, 6.0 Hz, 1H), 5.15 (t, J=5.5 Hz,1H), 5.00 (s, 2H), 4.65 (dt, J=5.4, 2.4 Hz, 1H), 4.12 (td, J=4.7, 2.2Hz, 1H), 3.02-2.88 (m, 1H), 2.84 (q, J=7.3 Hz, 2H), 2.61 (m, 1H), 1.35(s, 9H).

3′-O-tert-butyldithiomethyl-dATP (A5):N⁶-Benzoyl-3′-O-tert-butyldithiomethyl-2′-deoxyadenosine (A4, 50 mg,0.10 mmol), tetrabutylammonium pyrophosphate (180 mg, 0.33 mmol) and2-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one (44 mg, 0.22 mmol) weredried separately overnight under high vacuum at ambient temperature. Thetetrabutylammonium pyrophosphate was dissolved in dimethylformamide(DMF, 1 mL) under argon followed by addition of tributylamine (1 mL).This mixture was injected into the solution of2-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one in (DMF, 2 mL) underargon. After stirring for 1 h, the reaction mixture was added to thesolution of N⁶-Benzoyl-3′-O-tert-butyldithiomethyl-2′-deoxyadenosine andstirred further for 1 hour at room temperature. Iodine solution (0.02 Miodine/pyridine/water) was then injected into the reaction mixture untila permanent brown color was observed. After 10 min, water (30 mL) wasadded and the reaction mixture was stirred at room temperature for anadditional 2 hours. The resulting solution was extracted with ethylacetate. The aqueous layer was concentrated in vacuo to approximately 20mL, then concentrated NH₄OH (20 ml) was added and stirring continuedovernight at room temperature. The resulting mixture was concentratedunder vacuum and the residue was diluted with 5 ml of water. The crudemixture was then purified by anion exchange chromatography onDEAE-Sephadex A-25 at 4° C. using a gradient of TEAB (pH 8.0; 0.1-1.0M). The crude product was further purified by reverse-phase HPLC toafford A5, which was characterized by MALDI-TOF MS calc'd forC₁₅H₂₆N₅O₁₂P₃S₂: 625.4, found: 625.0.

N⁴-Benzoyl-3′-O-methylthiomethyl-5′-O-tert-butyldimethylsilyl-2′-deoxycytidine(C2): To a stirring solution ofN⁴-Benzoyl-5′-O-tert-butyldimethylsilyl-2′-deoxycytidine (C1, 1.5 g, 3.4mmol) in DMSO (6.5 mL) was added acetic acid (2.91 mL) and aceticanhydride (9.29 mL). The reaction mixture was stirred at roomtemperature for 2 days. Then the reaction mixture was added dropwise tosolution of sodium bicarbonate and extracted by ethyl acetate (50m1×3).The obtained crude product was purified by column chromatography (ethylacetate/hexane: 8:2) to give pure product C2 (1.26 g, 74%) as a whitesolid.¹H NMR (400 MHz, CDCl₃) δ8.43 (d, J=7.4 Hz, 1H), 7.92 (d, J=7.6Hz, 2H), 7.69-7.50 (m, 4H), 6.31 (t, J=6.1 Hz, 1H), 4.75-4.59 (m, 2H),4.51 (dt, J=6.2, 3.9 Hz, 1H), 4.20 (dt, J=3.7, 2.6 Hz, 1H), 4.01 (dd,J=11.4, 2.9 Hz, 1H), 3.86 (dd, J=11.4, 2.4 Hz, 1H), 2.72 (ddd, J=13.8,6.2, 4.1 Hz, 1H), 2.18 (s, 4H), 0.97 (s, 9H), 0.17 (d, J=3.9 Hz, 6H).HRMS (ESI⁺) calc'd for C₂₄H₃₅N₃O₅SSi [(M+H)⁺]: 506.2145, found:506.2146.

N⁴-Benzoyl-3′-O-ieri-butyldithiomethyl-5′-O-teri-butyldimethylsilyl-2′-deoxycytidine(C3):N⁴-Benzoyl-3′-O-methylthiomethyl-5′-O-tert-butyldimethylsilyl-2′-deoxycytidine(C2, 1.01 g, 2 mmol) was dissolved in anhydrous dichloromethane (8 mL),followed by addition of triethylamine (278 μL, 2 mmol) and molecularsieves (3 A, 1 g). The mixture was cooled in an ice bath after stirringat room temperature for 0.5 hour and then a solution of sulfurylchloride (161 μL, 2.2 mmol) in anhydrous dichloromethane (8 mL) wasadded dropwise. The ice bath was removed and the reaction mixture wasstirred further for 0.5 hour. Then potassium p-toluenethiosulfonate (678mg, 3 mmol) in anhydrous DMF (1 mL) was added to the mixture. Stirringwas continued at room temperature for an additional 1 hour followed byaddition of tert-butyl mercaptan (1 mL). The reaction mixture wasstirred at room temperature for 0.5 hour and quickly filtered. Thesolvent was removed under reduced pressure and the residue was dissolvedin ethyl acetate and washed in brine (3×50 mL). The combined organiclayers were dried over Na₂SO₄ and filtered. The filtrate wasconcentrated to dryness under reduced pressure and the residue of thedesired compound was purified by silica gel column chromatography usinga gradient of ethyl acetate-hexane from 3:7 (v/v) to 5:5 (v/v), yielding959 mg (83%) C3 as a white foam. ¹H NMR (400 MHz, CDCl₃) δ8.43 (d, J=7.4Hz, 1H), 7.92 (d, J=7.6 Hz, 2H), 7.69-7.50 (m, 4H), 6.31 (t, J=6.1 Hz,1H), 4.75-4.59 (m, 2H), 4.51 (dt, J=6.2, 3.9 Hz, 1H), 4.20 (dt, J=3.7,2.6 Hz, 1H), 4.01 (dd, J=11.4, 2.9 Hz, 1H), 3.86 (dd, J=11.4, 2.4 Hz,1H), 2.72 (ddd, J=13.8, 6.2, 4.1 Hz, 1H), 2.18 (s, 4H), 0.97 (s, 9H),0.17 (d, J=3.9 Hz, 6H), 0.10 (s, 2H). HRMS (ESI⁺) calc'd for:C₂₇H₄₁N₃O₅S₂Si [(M+Na)⁺]: 602.2155, found: 602.2147.

N⁴-Benzoyl-3′-O-tert-butyldithiomethyl-2′-deoxycytidine (C4) To astirring solution ofN⁴-Benzoyl-3′-O-tert-butyldithiomethyl-5′-O-tert-butyldimethylsilyl-2′-deoxycytidine(C3, 958 mg, 1.66 mmol) in a mixture of tetrahydrofuran (24 ml),tetrabutylammonium fluoride (1.0M, 2.48 mL) was added in small portions,and stirred at room temperature for 3 hours. The reaction mixture waspoured into a saturated sodium bicarbonate solution (50 mL) andextracted with ethyl acetate (3×50 mL). The combined organic layers weredried over Na₂SO₄ and filtered. The filtrate was concentrated to drynessunder reduced pressure and the residue of the desired compound waspurified by silica gel column chromatography using a gradient of ethylacetate-hexane from 5:5 (v/v—), affording 435 mg (56%) C4 as a solidwhite powder. ¹H NMR (400 MHz, Methanol-d₄) δ8.52 (d, J=7.5 Hz, 1H),8.04-7.96 (m, 2H), 7.71-7.60 (m, 2H), 7.61-7.51 (m, 2H), 6.28-6.19 (m,1H), 4.95-4.86 (m, 2H), 4.54 (dt, J=6.0, 3.0 Hz, 1H), 4.23 (q, J=3.4 Hz,1H)_(;) 3.92-3.76 (m, 2H), 2.70 (ddd, J=13.9, 6.0, 2.9 Hz, 1H), 2.25(ddd, J=13.6, 7.2, 6.2 Hz, 1H), 1.37 (s, 9H). HRMS (ESI⁺) calc'd forC₂₁H₂₇N₃O₅S₂[(M+Na)⁺]: 488.1290, found: 488.1297.

3′-O-tert-butyldithiomethyl-dCTP (C5):N⁴-Benzoyl-3′-O-tert-butyldithiomethyl-2′-deoxycytidine (C4, 50 mg, 0.11mmol), tetrabutylammonium pyrophosphate (180 mg, 0.33 mmol) and2-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one (44 mg, 0.22 mmol) weredried separately overnight under high vacuum at ambient temperature. Thetetrabutylammonium pyrophosphate was dissolved in dimethylformamide(DMF, 1 mL) under argon followed by addition of tributylamine (1 mL).This mixture was injected into the solution of2-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one in (DMF, 2 mL) underargon. After stirring for 1 h, the reaction mixture was added to thesolution of N⁴-benzoyl-3′-O-tert-butyldithiomethyl-2′-deoxycytidine andstirred further for 1 hour at room temperature. Iodine solution (0.02 Miodine/pyridine/water) was then injected into the reaction mixture untila permanent brown color was observed. After 10 min, water (30 mL) wasadded and the reaction mixture was stirred at room temperature for anadditional 2 hours. The resulting solution was extracted with ethylacetate. The aqueous layer was concentrated in vacuo to approximately 20mL, then concentrated NH₄OH (20 ml) was added and the mixture stirredovernight at room temperature. The resulting mixture was concentratedunder vacuum and the residue was diluted with 5 ml of water. The cntdemixture was then purified by anion exchange chromatography onDEAE-Sephadex A-25 at 4° C. using a gradient of TEAB (pH 8.0; 0.1-1.0M). The crude product was further purified by reverse-phase HPLC toafford C5. HRMS (ESI−) calc'd for C₁₄H₂₅N₃O₁₃P₃S₂[(M−H)⁻]: 600.0042,found: 600.0033.

Trimethyl(3-((methylthio)methoxy)prop-1-yn-1-yl)silane (2): To asolution of 3-trimethylsilanyl-prop-2-yn-1-ol (1, 1.28 g, 10 mmol) inDMSO (10 mL) acetic acid (2.6 mL, 45 mmol) and acetic anhydride (8.6 mL,90 mmol) were added with stirring. The reaction mixture was stirred atroom temperature until the reaction was complete (24 h), which wasmonitored by TLC. Then the mixture was added slowly to a solution ofsodium bicarbonate under vigorous stirring and extracted with ethylacetate (3×30 mL). The combined organic layers were dried over Na₂SO₄and filtered. The filtrate was concentrated to dryness under reducedpressure and the desired compound was purified by silica gel columnchromatography (ethyl acetate/hexane: 1:10) to give pure product 2 (0.97g, 67%): ¹H NMR (300 MHz, CDCl₃) δ: 4.75 (s, 2H), 4.28 (s, 2H), 2.16 (s,3H), 0.20 (s, 9H); ¹³C NMR (75 MHz, CDCl₃) 8:101.12, 92.07,74.04, 55.48,14.42, 0.18.

2,2,2-trifluoro-N-(2-mercapto-2-methylpropyl)acetamide (4):1-amino-2-methylpropane-2-thiol hydrochloride (3) (1.0 g, 7 mmol) wasmixed with pyridine (2 mL) in dried benzene (15 mL). At 0° C.,trifluoroacetic anhydride (1.30 mL, 9.2 mmol) was slowly added to thestirred mixture, and stirring was continued overnight at ambienttemperature. Careful addition of 0.5 M Na₂CO₃/H₂O was followed byextraction (EtOAc) of the aqueous layer and removal of volatiles fromthe combined organic layers under vacuum. Flash chromatography of theresidue (30% ethyl acetate/hexane) gave 4 (4.0 g, 88%): ¹H NMR (400 MHz,CDCl₃) 6.85(br, 1H), 3.45 (m, 2H), 1.69 (s, 1H), 1.41 (s, 6H).

S-(((3-(trimethylsilyl)prop-2-yn-1-yl)oxy)methyl)4-methylbenzenesulfonothioate (5):Trimethyl(3-((methylthio)methoxy)prop-1-yn-1-yl)silane (2, 1.0 g, 5.32mmol) was dissolved in anhydrous dichloromethane (10 mL), followed byaddition of cyclohexene (3.4 mL). The mixture was cooled in an ice bathand then a solution of sulfuryl chloride (0.47 mL, 5.85 mmol,) inanhydrous dichloromethane (3 mL) was added dropwise during 2 minutes.The ice bath was removed and the reaction mixture was stirred furtherfor 1 hour. Then potassium thiotosylate (1.44 g, 6.38 mmol) in anhydrousDMF (5 mL) was added to the mixture. Stirring was continued at roomtemperature for an additional 1 hour. After concentrating the solution,the residue was purified by silica gel column chromatography (ethylacetate/hexane: 10:1) to give pure product 5 (1.29 g, 74%): ¹H NMR (300MHz, CDCl₃) δ: 7.86 (d, J=8.4Hz, 2H), 7.32 (d, J=8.4Hz, 2H), 5.40 (s,2H), 4.03 (s, 2H), 2.46 (s, 3H), 0.19 (s, 9H).

2,2,2-trifluoro-N-(2-methyl-2-(((prop-2-yn-1-yloxy)methyl)disulfanyl)propyl)acetamide(7): Et₃N (0.3 mL) was added to a stirred mixture of2,2,2-trifluoro-N-(2-mercapto-2-methylpropyl)acetamide (4, 0.87 g, 4.32mmol) and S-4(3-(trimethylsilyl)prop-2-yn-1-yl)oxy)methyl)4-methylbenzenesulfonothioate (5, 1.29 g, 3.93 mmol) in anhydrousdichloromethanc (20 mL) at ambient temperature and stirring wascontinued for 0.5 hour. Then, tetrabutylammonium fluoride THF solution(1.0M, 5.89 mL, 5.89 mmol) was added. The reaction mixture was stirredat room temperature for ˜10 minutes, and volatiles were evaporated undervacuum. Flash chromatography of the residue gave2,2,2-trifluoro-N-(2-methyl-2-(((prop-2-yn-1-yloxy)methyl)disulfanyl)propyl)acetamide(7, 0.83 g, 70%): ¹H NMR (300 MHz, CDCl₃) δ: 7.22 (br, 1H), 4.87 (s,2H), 4.30 (d, J=2.4Hz, 2H), 3.45 (d, J=6.4Hz, 2H), 2.50 (s, J=2.4Hz,1H), 1.27 (s, 6H); ¹³C NMR (75 MHz, CDCl₃) 8:158.16, 118.26, 79.81,77.93, 76.27, 56.50, 50.93, 47.32, 25.30.

5-Iodo-5′-O-tert-butyldimethylsilyl-thymidine (T2): A mixture of5-iodo-2′-deoxythymidine (T1, 1 g, 2.8 mmol), tert-butyldimethylsilylchloride (453 mg, 3.0 mmol) and imidazole (199 mg, 3.0 mmol) wasdissolved in dry DMF(15 mL) and stirred at room temperature overnight.The reaction mixture was poured into ice water (200 mL) under stirringand the precipitate was collected by suction filtration, then washedwith water and hexane. The obtained crude product was purified by columnchromatography (dichloromethane/methanol: 20:1) to give5-iodo-5′-O-tert-butyldimethylsilyl-thymidine (T2, 1.152 g, 88%),¹H NMR(400 MHz, CDCl₃) δ8.19 (s, 1H), 8.12 (s, 1H), 6.36-6.27 (m, 1H), 4.51(dd, J=5.7, 2.9 Hz, 1H), 4.11 (q, J=2.5 Hz, 1H), 4.04-3.83 (m, 2H), 2.45(ddd, J=13.5, 5.7, 2.3 Hz, 1H), 2.14 (ddd, J=13.6, 8.0, 5.8 Hz, 1H),1.86 (d, J=3.5 Hz, 1H), 0.97 (s, 9H), 0.19 (d, J=6.7 Hz, 6H).

5-Iodo-5′-O-tert-butyldimethylsilyl-3′-O-methylthiomethyl-thymidine(T3): To a stirring solution of the5-iodo-5′-O-tert-butyldimethylsilyl-thymidine (T2, 1009 mg, 2.35 mmol)in DMSO (10 mL) was added acetic acid (3.0 mL) and acetic anhydride (8mL). The reaction mixture was stirred overnight at room temperature,then added dropwise to a saturated solution of sodium bicarbonate undervigorous stirring and extracted with ethyl acetate. The combined organiclayers were dried over Na₂SO₄ and filtered. The filtrate wasconcentrated to dryness under reduced pressure to give the crudecompound which was purified by column chromatography(dichloromethane/methanol: 30:1) to give pure product5-iodo-5¹-O-tert-butyldimethylsilyl-3′-O-methylthiomethyl-thymidine (T3,789 mg, 64%) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ8.53 (s, 1H),8.13 (s, 1H), 6.26 (dd, J=8.5, 5.5 Hz, 1H), 4.75-4.60 (m, 2H), 4.50 (dt,J=5.9, 1.8 Hz, 1H), 4.18 (q, =2.3 Hz, 1H), 3.89 (ddd, J=43.8, 11.4, 2.5Hz, 2H), 2.51 (ddd, J=13.5, 5.5, 1.7 Hz, 1H), 2.18 (s, 3H), 2.08-1.97(m, 1H), 0.98 (s, 9H), 0.20 (d, J=5.3 Hz, 6H).

5-Iodo-5′-O-tert-butyldimethylsilyl-3′-O-(tert-butyldithiomethyl)-2′-thymidine(T4):5-Iodo-5′-O-tert-butyldimethylsilyl-3′-O-methylthiomethyl-thymidine (T3,754 mg, 1.42 mmol) was dissolved in anhydrous dichloromethane (20 mL),followed by addition of triethylamine (0.3 mL) and molecular sieves (3Å, 2 g). The mixture was cooled in an ice bath after stirring at roomtemperature for 0.5 hour and then a solution of sulfuryl chloride (0.12mL, 1.50 mmol) in anhydrous dichloromethane (3 mL) was added dropwiseover 2 minutes. The ice bath was removed and the reaction mixture wasstirred for a further 0.5 hour. Then potassium p-toluenethiosulfonate(0.61 g, 2.25 mmol) in anhydrous DMF (3 mL) was added to the mixture.Stirring was continued at room temperature for an additional 1 hourfollowed by addition of tent-butyl mercaptan (1 mL). The reactionmixture was stirred at room temperature for 0.5 hour and quicklyfiltered through celite. The filter was washed with dichloromethane andthe organic fraction was concentrated. The residue was purified bysilica gel column chromatography (dichloromethane/methanol 30′) to givecompound T4. (561 mg, 66%). ¹H NMR (400 MHz, CDCl₃) δ9.11 (s, 1H), 8.12(s, 1H), 6.25 (dd, J=8.5, 5.4 Hz, 1H), 4.91 (d, J=11.2 Hz, 1H), 4.80 (d,J=11.2 Hz, 1H), 4.53 (dt, J=6.0, 1.7 Hz, 1H), 4.22 (q, J=2.2 Hz, 1H),4.00-3.80 (m, 2H), 2.59-2.45 (m, 1H), 2.02 (ddd, J=13.6, 8.5, 5.9 Hz,1H), 1.36 (s, 9H), 0.98 (s, 9H), 0.19 (d, J=4.9 Hz, 6H).

Compound T5: Under nitrogen, a mixture of T4 (501 mg, 0.83 mmol), CuI(20 mg, 0.11 mmol) and triethylamine (0.30 mL) in thy DMF (5 mL) wasstirred at room temperature for 5 min followed by the addition of DTMlinker 7 (277 mg, 0.91 mmol), and Pd(0) (150 mg, 0.13 mmol). Afterstirring at room temperature in the dark overnight, the reaction mixturewas added dropwisc into brine (200 mL) under vigorous stirring andextracted with ethyl acetate. The combined organic layers were driedover Na₂SO₄ and filtered. The filtrate was concentrated to dryness underreduced pressure to give the crude compound T5. MS (APCI—) calc'd forC₃₀H₄₈F₃N₃O₇S₄Si: 776.0, found: 774.5.

Compound T6: Without isolation, the crude compound T5 was dissolved inTHF (10 mL) followed by the addition of TBAF THF solution (1.0M, 1.0 mL,1.0 mmol). The mixture was stirred overnight at room temperature. Then,the mixture was concentrated in vacuo, saturated NaHCO₃ solution (50 mL)was added and the mixture was extracted with dichloromethane. Theorganic layer was dried over anhydrous Na₂SO₄, filtered, concentratedand the obtained crude mixture was purified by flash columnchromatography (dichloromethane/methanol: 20:1) to give compound T6 (161mg, 29% from compound T4). ¹H NMR (400 MHz, CDCl₃) δ8.50 (s, 1H), 8.11(s, 1H), 7.45 (s, 1H), 6.22 (dd, J=7.6, 5.9 Hz, 1H), 4.93-4.85 (m, 3H),4.81 (d, J=11.2 Hz, 1H), 4.58 (dt, J=6.0, 2.9 Hz, 1H), 4.52 (s, 2H),4.20 (q, J=2.7 Hz, 1H), 4.02 (ddd, J=11.9, 4.4, 2.6 Hz, 1H), 3.89 (ddd,J=11.8, 5.0, 2.7 Hz, 1H), 3.54-3.47 (m, 2H), 2.76 (t, J=4.7 Hz, 1H),2.54 (ddd, J=13.8, 6.0, 2.8 Hz, 1H), 2.25 (ddd, J=13.8, 7.6, 6.4 Hz,1H), 1.37 (s, 9H), 1.34 (s, 6H). ¹³C NMR (75 MHz, CDCl₃) δ162.00,158.47, 157.99, 149.59, 144.64, 118.29, 114.47, 99.27, 87.93, 86.84,85.66, 81.24, 80.62, 78.95, 77.86, 77.70, 77.43, 77.01, 62.54, 57.67,53.82, 50.82, 48.03, 47.41, 38.52, 35.04, 31.95, 30.28, 29.43, 25.97,25.66, 23.01, 21.08, 14.48, 11.79. MS (APCI⁺) calc'd for C₂₄H₃₄F₃N₃O₇S₄:661.8, found: 661.4.

3′-O-DTM-dUTP-5—SS-NH₂ (Compound T7): Compound T6 (50 mg, 76 μmol),tetrabutylammonium pyrophosphate (100 mg, 0.18 mmol) and2-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one (22 mg, 0.11 mmol) weredried separately overnight under high vacuum at ambient temperature. Thetetrabutylammonium pyrophosphate was dissolved in dimethylformamide(DMF, 1 mL) under argon followed by addition of tributylamine (1 mL).The mixture was injected into the solution of2-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one in (DMF, 2 mL) underargon. After stirring for 1 h, the reaction mixture was added to thesolution of T6 in DMF and stirred further for 1 hour at roomtemperature. Iodine solution (0.02 M iodine/pyridine/water) was theninjected into the reaction mixture until a permanent brown color wasobserved. After 10 min, water (30 mL) was added and the reaction mixturewas stirred at room temperature for an additional 2 hours. The resultingsolution was extracted with ethyl acetate. The aqueous layer wasconcentrated in vacuo to approximately 20 mL, then concentrated NH₄OH(20 ml) was added and stirring continued overnight at room temperature.The resulting mixture was concentrated under vacuum and the residue wasdiluted with 5 ml of water. The crude mixture was then purified by anionexchange chromatography with DEAE-Sephadex A-25 at 4° C. using agradient of TEAB (pH 8.0; 0.1-1.0 M). The crude product was furtherpurified by reverse-phase HPLC to afford T7, which was characterized byMALDI-TOF MS: calc'd for C₂₂H₃₈N₃O₁₅P₃S₄: 805.0, found: 809.1.

3′-O-DTM-dUTP-5—SS-R6G (Compound T8): To a stirred solution of Rhodamine6G-NHS ester (2 mg, 3.6 umol) in DMF (0.2 ml), 3′-O-DTM-dUTP-5—SS-NH₂(compound T7, 1.5 μmol) in NaHCO₃/Na₂CO₃ buffer (pH 8.9, 0.1 M, 0.3 ml)was added. The reaction mixture was stirred at room temperature for 3 hwith exclusion of light. The reaction mixture was purified by anionexchange chromatography on DEAE-Sephadex A-25 at 4° C. using a gradientof TEAB (pH 8.0; 0.1-1.0 M). The crude product was further purified onreverse-phase HPLC to afford compound T8, which was characterized byMALDI-TOF MS: calc'd for C₄₉H₃₄F₃N₃O₇S₄: 1249.2, found: 1248.9.

N⁴-DMF-5-iodo-5′-O-tert-butyldimethylsilyl-2′-deoxycytidine (C2): Amixture of 5-iodo-2′-deoxycytidine (C1, 1 g, 2.8 mmol) ,tert-butyldimethylsilyl chloride (450 mg, 3.0 mmol) and imidazole (200mg, 3.0 mmol) was dissolved in dry DMF (15 mL) and stirred overnight atroom temperature. After this period, the solvent was removed and theresidue was added to N,N-dimethylformamide dimethyl acetal (1.5 mL) indry DMF (10 mL). Stirring was continued at room temperature for anadditional 10 hours, then the reaction mixture was poured into ice water(200 mL) under stirring, The precipitate was collected by filtration,and washed with water and hexane. The obtained crude product waspurified by column chromatography (dichloromethane/methanol⁻20:1) togive N⁴-DMF-5-iodo-5′-O-tert-butyldimethylsilyl-2′-deoxycytidine (C2,1.02 g, 70%). ¹H NMR (400 MHz, CDCl₃) δ8.74-8.69 (m, 1H), 8.27 (s, 1H),6.37 (dd, =7.8, 5.6 Hz, 1H), 4.46 (dt, J=5.9, 2.3 Hz, 1H), 4.16 (q,J=2.6 Hz, 1H), 3.94 (dd, J=11.3, 2.8 Hz, 1H), 3.84 (dd, J=11.3, 2.7 Hz,1H), 3.22 (d, J=0.8 Hz, 3H), 3.19 (d, =0.5 Hz, 3H), 2.69 (ddd, J=13.5,5.7, 2.4 Hz, 1H), 2.05 (ddd, J=13.5, 7.8, 5.7 Hz, 1H), 0.93 (s, 9H),0.15 (d, J=8.9 Hz, 6H). MS (APCI⁺) calc'd for C₁₈H₃₁IN₄O₄Si: 522.5,found: 522.5.

N⁴-DMF-5-iodo-5′-O-terl-butyldimethylsilyl-3′-O-methylthiomethyl-2′-deoxycytidine(C3): To a stirring solution of theN⁴-DMF-5-iodo-5′-O-tort-butyldimethylsilyl-2′-deoxycytidine (C2, 1.02 g,2.29 mmol) in DMSO (10 mL) was added acetic acid (2.3 mL) and aceticanhydride (6.1 mL). The reaction mixture was stirred at room temperatureovernight. Then the reaction mixture was added dropwise to a saturatedsolution of sodium bicarbonate under vigorous stirring and theprecipitate was collected by suction filtration, washed with water andhexane. The obtained crude product was purified by column chromatography(dichloromethane/methanol: 30:1) to give pure productN⁴-DMF-5-iodo-5′-O-tert-butyldimethylsilyl-3′-O-methylthiomethyl-2′-deoxycytidineas a white solid (C3, 1.05 mg, 79%). ¹H NMR (400 MHz, CDCl₃) δ8.4 (s,1H), 8.22 (s, 1H), 6.28 (dd, J=7.9, 5.6 Hz, 1H), 4.70 (d, J=11.6 Hz,1H), 4.61 (d, J=11.6 Hz, 1H), 4.48 (dt, J=6.1, 2.3 Hz, 1H), 4.17 (q,J=2.7 Hz, 1H), 3.97-3.88 (m, 1H), 3.82 (dd, J=11.2, 2.8 Hz, 1H), 3.21(ddd, J=17.7, 2.0, 0.6 Hz, 7H), 2.68 (ddd, J=13.6, 5.7, 2.1 Hz, 1H),2.16 (s, 3H), 1.97 (ddd, J=13.8, 7.9, 6.1 Hz, 1H), 0.96 (s, 9H), 0.17(d, J=6.1 Hz, 6H). MS (APCI⁺) calc'd for C₂₀H₃₅IN₄O₄SSi: 582.5, found:582.4.

N⁴-DMF-5-iodo-5′-O-tert-butyldimethylsilyl-3′-O-(len-butyldithiomethyl)-2′-deoxycytidine(C4):N⁴-DMF-5-iodo-5′-O-tert-butyldimethylsilyl-3′-O-methylthiomethyl-2′-deoxycytidine(C3, 1.05 g, 1.81 mmol) was dissolved in anhydrous dichloromethane (20mL), followed by addition of triethylamine (0.3 mL) and molecular sieves(3 Å, 2 g). The mixture was cooled in an ice bath, stirred at roomtemperature for 0.5 hour and then a solution of sulfuryl chloride (0.16mL, 1.99 mmol) in anhydrous dichloromethane (3 mL) was added dropwiseover 2 minutes. The ice bath was removed and the reaction mixture wasstirred for a further 0.5 hour. Then potassium p-toluenethiosulfonate(614 mg, 2.71 mmol) in anhydrous DMF (3 mL) was added to the mixture.Stirring was continued at room temperature for an additional 1 hourfollowed by addition of tert-butyl mercaptan (1 mL). The reactionmixture was stirred at room temperature for 0.5 hour and quicklyfiltered through celite. The filter was washed with dichloromethane andthe organic fraction was concentrated. The residue was purified bysilica gel column chromatography (dichloromethanc/mcthanol: 30:1) togive compound C4 (648 mg, 53%). ¹H NMR (400 MHz, CDCl₃) δ8.77-8.71 (m,1H), 8.23 (s, 1H), 6.28 (dd, J=8.0, 5.6 Hz, 1H), 4.92 (d, J=11.2 Hz,1H), 4.77 (d. J=11.2 Hz, 1H), 4.52 (dt, J=5.9, 1.9 Hz, 1H), 4.21 (q,J=2.5 Hz, 1H), 3.95 (dd, J=11.3, 2.7 Hz, 1H), 3.84 (dd, J=11.3, 2.6 Hz,1H), 3.23 (d, J=0.7 Hz, 3H), 3.19 (d, J=0.5 Hz, 3H), 2.72 (ddd, J=13.7,5.6, 1.9 Hz, 1H), 1.96 (ddd, J=13.9, 8.1, 6.0 Hz, 1H), 1.35 (s, 9H),0.96 (s, 9H), 0.18 (d, J=5.9 Hz, 6H); MS (APO): calc'd forC₂₃H₄₁IN₄O₄S₂Si: 656.7, found: 656.5.

Compound C5: Under nitrogen, a mixture ofN⁴-DMF-5-iodo-5′-O-tert-butyldimethylsilyl-3′-O-(tert-butyldithiomethyl)-2′-deoxycytidine(C4, 420 mg, 0.64 mmol 0.375 mmol), CO (20 mg, 0.11 mmol) andtriethylamine (0.30 mL) in dry DMF (5 mL) was stirred at roomtemperature for 5 min followed by the addition of DTM linker 7 (213 mg,0.70 mmol), and Pd(0) (150 mg, 0.13 mmol). After stirring at roomtemperature in the dark overnight, the reaction mixture was addeddropwise into brine (200 mL) under vigorous stirring and extracted withethyl acetate. The combined organic layers were dried over Na₂SO₄ andfiltered. The filtrate was concentrated to dryness under reducedpressure to give the crude compound C5. MS (APCI⁺) calc'd forC₃₃H₅₄F₃N₅O₆S₄Si: 830.1, found: 829.2.

Compound C6: Without isolation, the crude compound C5 was dissolved inTHF (10 mL) followed by the addition of TBAF fluoride THF solution(1.0M, 1.0 mL, 1.0 mmol). The mixture was stirred for 2 h at roomtemperature. Then, the mixture was concentrated in vacuo, saturatedNaHCO₃ solution (50 mL) was added and the mixture was extracted withdichloromethane. The organic layer was dried over anhydrous Na₂SO₄,filtered, concentrated and the obtained crude mixture was purified byflash column chromatography (dichloromethane/methanol: 20:1) to givecompound C6 (146 mg, 32% from compound C4). ¹H NMR (400 MHz, CDCl₃)δ8.77 (s, 1H), 8.09 (s, 1H), 7.44 (d, J=7.3 Hz, 1H), 6.11 (t, J=6.6 Hz,1H), 4.95 (s, 2H), 4.87 (d, J=11.1 Hz, 1H), 4.76 (d, J=11.1 Hz, 1H),4.60-4.48 (m. 3H), 4.18 (q, J=3.0 Hz, 1H), 3.98 (dd, J=12.1, 2.7 Hz,1H), 3.84 (dd, J=12.0, 3.2 Hz, 1H), 3.47 (d, J=6.4 Hz, 2H), 3.20-3.15(m, 6H), 2.96 (s, 1H), 2.88 (d, J=0.6 Hz, 1H), 2.59 (ddd, J=13.8, 6.1,3.2 Hz, 1H), 2.35 (dt, J=13.7, 6.8 Hz, 1H), 1.33 (s, 9H), 1.31 (s, 6H).¹³C NMR (75 MHz, CDCl₃) δ 170.96, 162.94, 159.08, 154.94, 148.67,146.84, 98.35, 89.16, 86.47, 85.90, 82.09, 81.22, 79.85, 77.65, 62.66,57.65, 50.94, 47.92, 47.31, 41.87, 41.81, 38.42, 38.03, 36.86, 35.76,31.82, 30.29, 26.01. MS (APCI⁺): calc'd for C₂₇H₄₀F₃N₅O₆S₄: 715.9,found:715.9.

3′-O-DTM-dCTP-5—SS-NH₂ (Compound C7). Compound C6 (50 mg, 0.07 mmol),tetrabutylammonium pyrophosphate (99 mg, 0.18 mmol) and2-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one (22 mg, 0.11 mmol) weredried separately overnight under high vacuum at ambient temperature. Thetetrabutylammonium pyrophosphate was dissolved in dimethylformamide(DMF, 1 mL) under argon followed by addition of tributylamine (1 mL).The mixture was injected into the solution of2-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one in (DMF, 2 mL) underargon. After stirring for 1 h, the reaction mixture was added to thesolution of C6 in DMF and stirred further for 1 hour at roomtemperature. Iodine solution (0.02 M iodine/pyridine/water) was theninjected into the reaction mixture until a permanent brown color wasobserved. After 10 min, water (30 mL) was added and the reaction mixturewas stirred at room temperature for an additional 2 hours. The resultingsolution was extracted with ethyl acetate. The aqueous layer wasconcentrated in vacuo to approximately 20 mL, then concentrated NH₄OH(20 ml) was added and the mixture stirred overnight at room temperature.The resulting mixture was concentrated under vacuum and the residue wasdiluted with 5 ml of water. The crude mixture was then purified by anionexchange chromatography on DEAE-Sephadex A-25 at 4° C. using a gradientof TEAB (pH 8.0; 0.1-1.0 M). The crude product was further purified byreverse-phase HPLC to afford C7, which was characterized by MALDI-TOFMS: calc'd for C₂₂H₃₉N₄O₁₄P₃S₄: 804.0, found: 807.5.

3′-O-DTM-S —SS-Alexa488-dCTP (Compound C8). To a stirred solution ofAlexa488—NHS ester (2 mg, 3.1μmol) in DMF (0.2 ml),3′-O-DTM-dCTP-5—SS-NH₂ (compound C7, 3.0 μmol) in NaHCO₃/Na₂CO₃ buffer(pH 8.9, 0.1 M, 0.3 ml) was added. The reaction mixture was stirred atroom temperature for 3 h with exclusion of light. The reaction mixturewas purified by anion exchange chromatography on DEAE-Sephadex A-25 at4° C. using a gradient of TEAB (pH 8.0; 0.1-1.0 M). The crude productwas further purified on reverse-phase HPLC to afford Compound C8, whichwas characterized by MALDI-TOF MS: calc'd for C₄₃H₄₉N₆O₂₄P₃S₆ ²⁻:1319.2, found: 1325.1.

N⁴-DMF-7-deaza-7-iodo-5′-O-tert-butyldimethylsilyl-2′-deoxyadenosine(A2): A mixture of 7-deaza-7-iodo-2′-deoxyadenosine (A1, 1 g, 2.66 mmol), tert-butyldimethylsilyl chloride (440 mg, 2.9 mmol) and imidazole (200mg, 3.0 mmol) was dissolved in dry DMF(15 mL) and stirred overnight atroom temperature. After this period, the solvent was removed and theresidue was added to N,N-dimethylformamide dimethyl acetal (1.5 mL) indry DMF (10 mL). Stirring was continued at room temperature for anadditional 10 hours, then the reaction mixture was poured into ice water(200 mL) under stirring. The precipitate was collected by suctionfiltration, then washed with water and hexane. The obtained crudeproduct was purified by column chromatography (dichloromethane/methanol:20:1) to giveN⁴-DMF-7-deaza-7-iodo-5′-O-tert-butyldimethylsilyl-2′-deoxyadenosine(A2, 1145 mg, 79%). MS (APCI⁺) calc'd for C₂₀H₃₂IN₅O₃Si: 545.5, found:545.7.

N⁴-DMF-7-deaza-7-iodo-5′-O-tert-butyldimethylsilyl-3′-O-methylthiomethyl-2′-deoxyadenosine(A3): To a stirring solution of theN⁴-DMF-7-deaza-7-iodo-5′-O-tert-butyldimethylsilyl-2′-deoxyadenosine(A2, 1324 mg, 2.43 mmol) in DMSO (10 mL) was added acetic acid (3 mL)and acetic anhydride (8 mL). The reaction mixture was stirred at roomtemperature overnight. Then the reaction mixture was added dropwise to asaturated solution of sodium bicarbonate under vigorous stirring and theprecipitate was collected by suction filtration, and washed with waterand hexane. The obtained crude product was purified by columnchromatography (dichloromethane/methanol: 30:1) to give pure productN⁴-DMF-7-deaza-7-iodo-5′-O-tert-butyldimethylsilyl-3′-O-methylthiomethyl-2′-deoxyadenosineas a white solid (A3, 956 mg, 65%). ¹H NMR (400 MHz, CDCl₃) δ8.82-8.77(m, 1H), 8.45 (s, 1H), 7.48 (s, 1H), 6.70 (dd, J=7.7, 6.2 Hz, 1H),4.76-4.64 (m, 2H), 4.64-4.55 (m, 1H), 4.14 (td, J=3.7, 2.4 Hz, 1H),3.86-3.80 (m, 2H), 3.32 (d, .I=0.6 Hz, 3H), 3.22-3.17 (m, 3H), 2.54-2.42(m, 2H), 2.19 (s, 3H), 0.97 (s, 9H), 0.15 (d, J=6.3 Hz, 6H). MS (APCI⁺)calc'd for C22H361N5O3SSi: 605.6, found: 605.1.

N⁴-DMF-7-deaza-7-iodo-5′-O-tert-butyldimethylsilyl-3′-O-(tert-butyldithiomethyl)-2′-deoxyadenosine(A4):N⁴-DMF-7-deaza-7-iodo-5′-O-tert-butyldimethylsilyl-3′-O-methylthiomethyl-2′-deoxyadenosine(A3, 900 mg, 1.48 mmol) was dissolved in anhydrous dichloromethane (20mL), followed by addition of triethylamine (0.3 mL) and molecular sieves(3 A, 2 g). The mixture was cooled in an ice bath, stirred at roomtemperature for 0.5 hour and then a solution of sulfuryl chloride (0.13mL, 1.63 mmol) in anhydrous dichloromethane (3 mL) was added dropwiseover 2 minutes. The ice bath was removed and the reaction mixture wasstirred for a further 0.5 hour. Then potassium p-toluenethiosulfonate(509 mg, 2.22 mmol) in anhydrous DMF (3 mL) was added to the mixture.Stirring was continued at room temperature for an additional 1 hourfollowed by addition of tert-butyl mercaptan (1 mL). The reactionmixture was stirred at room temperature for 0.5 hour and quicklyfiltered through celite. The filter was washed with dichloromethane andthe organic fraction was concentrated. The residue was purified bysilica gel column chromatography (dichloromethane/methanol: 30:1) togiveN⁴-DMF-7-deaza-7-iodo-5′-O-tert-butyldimethylsilyl-3′-O-(tert-butyldithiomethyl)-2′-deoxyadenosine(A4, 733 mg, 73%). ¹H NMR (400 MHz, CDCl₃) δ8.82-8.77 (m, 1H), 8.45 (s,1H), 7.50 (s, 1H), 6.69 (dd, J=8.0, 6.0 Hz, 1H), 4.95-4.80 (m, 2H), 4.63(dt, J=5.3, 2.5 Hz, 1H), 4.17 (td, J=3.5, 2.3 Hz, 1H), 3.85 (dd, J=3.5,1.2 Hz, 2H), 3.32 (d, J=0.6 Hz, 3H), 3.19 (s, 3H), 2.58-2.41 (m, 2H),1.36 (s, 9H), 0.98 (s, 9H), 0.16 (d, J=6.0 Hz, 6H). MS (APCI⁺) calc'dfor C₂₅H₄₂IN₅O₃S₂Si: 679.7, found:679.4

Compound A5: Under nitrogen, a mixture ofN⁴-DMF-7-deaza-7-iodo-5′-O-tert-butyldimethylsilyl-3′-O-(tert-butyldithiomethyl)-2′-deoxyadenosine(A4, 444 mg, 0.65 mmol), CuI (20 mg, 0.11 mmol) and triethylamine (0.30mL) in dry DMF (5 mL) was stirred at room temperature for 5 min followedby the addition of DTM linker 7 (310 mg, 1.02 mmol), and Pd(0) (150 mg,0.13 mmol). After stirring at room temperature in the dark overnight,the reaction mixture was added dropwise into brine (200 mL) undervigorous stirring and extracted with ethyl acetate. The combined organiclayers were dried over Na₂SO₄ and filtered. The filtrate wasconcentrated to dryness under reduced pressure to give the crudecompound AS. MS (APCI⁺) calc'd for C₃₅H₅₅F₃N₆O₅S₄Si: 853.2, found: 853.1

Compound A6: Without isolation, the crude compound AS was dissolved inTHF (10 mL) followed by the addition of TBAF fluoride THF solution(1.0M, 1.0 mL, 1.0 mmol). The mixture was stirred at room temperaturefor 2 h. Then the mixture was concentrated in vacuo, saturated NaHCO₃solution (50 mL) was added and the mixture was extracted withdichloromethane. The organic layer was dried over anhydrous Na₂SO₄,filtered, concentrated and the obtained crude mixture was purified byflash column chromatography (dichloromethane/methanol: 20:1) to givecompound A6 (105 mg, 22% from compound A4). ¹H NMR (400 MHz, CDCl₃)δ8.81 (d, J=6.7 Hz, 1H), 8.41 (d, J=9.7 Hz, 1H), 7.28 (s, 1H), 6.12 (dt,J=12.8, 6.5 Hz, 1H), 4.98 (s, 2H), 4.88 (d, J=3.3 Hz, 2H), 4.73 (d,J=5.4 Hz, 1H), 4.58 (s, 2H), 4.33 (d, J=4.9 Hz, 1H), 4.01 (dt, J=12.9,3.1 Hz, 1H), 3.83 (d, J=8.2 Hz, 1H), 3.48 (d, J=6.4 Hz, 2H), 3.23 (d,J=17.2 Hz, 6H), 3.13-3.00 (m, 1H), 2.40 (dt, J=13.3, 6.6 Hz, 1H), 1.36(s, 9H), 1.34 (s, 6H). ¹³C NMR (75 MHz, CDCl₃) 6 162.23, 158.17, 157.08,152.13, 151.48, 150.16, 131.12, 113.22, 96.29, 90.20, 86.94, 84.09,82.11, 81.09, 80.16, 79.55, 64.07, 57.57, 51.02, 47.91, 47.28, 41.36,37.94, 35.91, 35.34, 30.33, 26.01. MS (APCP): calc'd for C₂₉H₄₁F₃N₆O₅S₄:738.9, found:737.2.

3′-O-DTM-7-deaza-dATP-7—SS-NH₂ (Compound A7): Compound A6 (40 mg, 54μmol), tetrabutylammonium pyrophosphate (60 mg, 108 pimol) and2-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one (22 mg, 110 μmol) weredried separately over night under high vacuum at ambient temperature.The tetrabutylammonium pyrophosphate was dissolved in dimethylformamide(DMF, 1 mL) under argon followed by the addition of tributylamine (1mL). The mixture was injected into the solution of2-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one in (DMF, 2 mL) underargon. After stirring for 1 h, the reaction mixture was added to asolution of A6 in DMF and stirred for a further 1 hour at roomtemperature. Iodine solution (0.02 M iodine/pyridine/water) was theninjected into the reaction mixture until a permanent brown color wasobserved. After 10 min, water (30 mL) was added and the reaction mixturewas stirred at room temperature for an additional 2 hours. The resultingsolution was extracted with ethyl acetate. The aqueous layer wasconcentrated in vacuo to approximately 20 mL, then concentrated NH₄OH(20 ml) was added and stirring continued overnight at room temperature.The resulting mixture was concentrated under vacuum and the residue wasdiluted with 5 ml of water. The crude mixture was then purified by anionexchange chromatography on DEAE-Sephadex A-25 at 4° C. using a gradientof TEAB (pH 8.0; 0.1-1.0 M). The crude product was further purified byreverse-phase HPLC to afford A7, which was characterized by MALDI-TOFMS: calc'd for C₂₄H₄₀N₅O₁₃P₃S₄: 827.0, found: 830.1.

3′-O-DTM-7-deaza-dATP-7—SS-ROX (Compound A8): To a stirred solution ofROX-NHS ester (2 mg, 3.2 μmol) in DMF (0.2 ml),3′-O-DTM-7-deaza-dATP-7—SS-NH2 (compound A7, 3.0 mol) in NaHCO3/Na₂CO₃buffer (pH 8.9, 0.1 M, 0.3 ml) was added. The reaction mixture wasstirred at room temperature for 3 h with exclusion of light. Thereaction mixture was purified by anion exchange chromatography onDEAE-Sephadex A-25 at 4° C. using a gradient of TEAB (pH 8.0; 0.1-1.0M). The crude product was further purified by reverse-phase HPLC toafford Compound A8, which was characterized by MALDI-TOF MS: calc'd forC₅₇H₆₉N₇O₁₇P₃S₄ ⁺: 1344.3, found: 1345.5.

N⁴-DMF-7-deaza-7-iodo-5′-O-tert-butyldimethylsilyl-2′-deoxyguanosine(G2): A mixture of 7-deaza-7-iodo-2′-deoxyguanosine (G1, 1 g, 2.55mmol), tert-butyldimethylsilyl chloride (420 mg, 2.8 mmol) and imidazole(202 mg, 3.0 mmol) was dissolved in dry DMF (15 mL) and stirredovernight at room temperature. After this period, the solvent wasremoved and the residue was added to N,N-dimethylformamide dimethylacetal (1.5 mL) in dry DMF (10 mL). Stirring was continued at roomtemperature for an additional 10 hours, then the reaction mixture waspoured into ice water (200 mL) under stirring and the precipitate wascollected by suction filtration, and washed with water and hexane. Theobtained crude product was purified by column chromatography(dichloromethane/methanol: 20:1) to giveN′-DMF-7-deaza-7-iodo-5′-O-tert-butyldimethylsilyl-2′-deoxyguanosine(G2, 1.07 g, 77%). ¹H NMR (400 MHz, CDCl₃) δ8.63 (s, 1H), 8.60 (s, 1H),7.11 (s, 1H), 6.63 (dd, J=7.5, 6.2 Hz, 1H), 4.62 (s, 1H), 4.03 (dt,J=4.7, 3.1 Hz, 1H), 3.88 (dd, J=10.8, 3.2 Hz, 1H), 3.79 (dd, J=10.8, 4.7Hz, 1H), 3.52 (s, 1H), 3.19 (s, 3H), 3.09 (d, J=0.6 Hz, 3H), 2.53-2.34(m, 2H), 0.97 (s, 9H), 0.21-0.10 (m, 6H).

N⁴-DMF-7-deaza-7-iodo-5′-O-tert-butyldimethylsily⁻1-3′-O-methylthiomethyl-2′-deoxyguanosine(G3): To a stirring solution of theN⁴-DMF-7-deaza-7-iodo-5′-O-tert-butyldimethylsilyl-2′-deoxyguanosine(G2, 950 mg, 1.74 mmol) in DMSO (10 mL) was added acetic acid (3 mL) andacetic anhydride (8 mL). The reaction mixture was stirred at roomtemperature overnight. The reaction mixture was added dropwise to asaturated solution of sodium bicarbonate under vigorous stirring and theprecipitate was collected by suction filtration, and washed with waterand hexane. The obtained crude product was purified by columnchromatography (dichloromethane/methanol: 30:1) to give pure productN⁴-DMF-7-deaza-7-iodo-5′-O-tert-butyldimethylsilyl-3′-O-methylthiomethyl-2′-deoxyguanosineas a white solid (G3, 756 mg, 70%). ¹H NMR (400 MHz, CDCl₃) δ9.66 (s,1H), 8.62 (d, J=0.9 Hz, 1H), 7.11 (s, 1H), 6.56 (dd, J=8.1, 6.2 Hz, 1H),4.76-4.64 (m, 2H), 4.59 (dt, J=5.2, 2.7 Hz, 1H), 4.11 (ddd, J=4.4, 3.1,2.2 Hz, 1H), 3.88-3.70 (m, 2H), 3.18 (s, 3H), 3.16-3.08 (m, 3H),2.48-2.32 (m, 2H), 2.19 (s, 3H), 0.97 (s, 9H), 0.15 (d, J=5.9 Hz, 6H).

N⁴-DMF-7-deaza-7-iodo-5′-O-tert-butyldimethylsily⁻1-3′-O-(tert-butyldithiomethyl)-2′-deoxyguanosine(G4):N⁴-DMF-7-deaza-7-iodo-5′-O-tert-butyldimethylsilyl-3′-O-methylthiomethyl-2′-deoxyguanosine(G3, 731 mg, 1.18 mmol) was dissolved in anhydrous dichloromethane (20mL), followed by addition of triethylamine (0.2 mL) and molecular sieves(3 Å, 2 g). After stirring at room temperature for 0.5 hour and coolingin an ice bath, a solution of sulfuryl chloride (0.11 mL, 1.33 mmol) inanhydrous dichloromethane (3 mL) was added dropwise over 2 minutes. Theice bath was removed and the reaction mixture was stirred for a further0.5 hour. Then potassium p-toluenethiosulfonate (417 mg, 1.82 mmol) inanhydrous DMF (2 mL) was added to the mixture. Stirring was continued atroom temperature for an additional 1 hour followed by the addition oftert-butyl mercaptan (1 mL). The reaction mixture was stirred at roomtemperature for 0.5 hour and quickly filtered through celite. The filterwas washed with dichloromethane and the organic fraction wasconcentrated. The residue was purified by silica gel columnchromatography (dichloromethane/methanol: 30:1) to giveN⁴-DMF-7-deaza-7-iodo-5′-O-tert-butyldimethylsilyl-3′-O-(tert-butyldithiomethyl)-2′-deoxyguanosine(G4, 508 mg, 62%). ¹H NMR (400 MHz, CDCl₃) δ9.11 (s, 1H), 8.64 (t, J 0.6Hz, 1H), 7.12 (s, 1H), 6.56 (dd, J=8.4, 5.9 Hz, 1H), 4.94-4.83 (m, 2H),4.59 (dt, J=5.8, 2.2 Hz, 1H), 4.19-4.12 (m, 1H), 3.88-3.75 (m, 2H), 3.20(s, 3H), 3.11 (d, J=0.7 Hz, 3H), 2.52-2.34 (m, 2H), 1.42-1.30 (m, 9H),0.98 (s, 9H), 0.16 (d, J=6.0 Hz, 6H).

Compound G5: Under nitrogen, a mixture ofN⁴-DMF-7-deaza-7-iodo-5′-O-tert-butyldimethylsilyl-3′-O-(tert-butyldithiomethyl)-2′-deoxyguanosine(G4, 471 mg, 0.68 mmol), CuI (20 mg, 0.11 mmol) and triethylamine (0.30mL) in dry DMF (5 mL) was stirred at room temperature for 5 min followedby the addition of DTM linker 7 (300 mg, 0.99 mmol), and Pd(0) (150 mg,0.13 mmol). After stirring at room temperature in the dark overnight,the reaction mixture was added dropwise into brine (200 mL) undervigorous stirring and extracted with ethyl acetate. The combined organiclayers were dried over Na₂SO₄ and filtered. The filtrate wasconcentrated to dryness under reduced pressure to give crude compoundG5.

Compound G6: Without isolation, the crude compound G5 was dissolved inTHF (10 mL) followed by the addition of TBAF fluoride THF solution(1.0M, 1.0 mL, 1.0 mmol). The mixture was stirred at room temperaturefor 2 h. Then, the mixture was concentrated in vacuo, saturated NaHCO₃solution (50 mL) was added and the mixture was extracted withdichloromethane. The organic layer was dried over anhydrous Na₂SO₄,filtered, concentrated and the obtained crude mixture was purified byflash column chromatography (dichloromethane/methanol: 20:1) to givecompound G6 (121 mg, 24% from compound G4). ¹H NMR (400 MHz, CDCl₃)δ8.92 (s, 1H), 8.52 (s, 1H), 7.66 (s, 1H), 7.01 (s, 1H), 6.19 (t, J=7.2Hz, 1H), 4.99 (s, 2H), 4.87 (q, J=11.1 Hz, 3H), 4.67 (s, 1H), 4.56 (s,2H), 4.22 (s, 1H), 4.06-3.99 (m, 1H), 3.91 (d, J=12.1 Hz, 1H), 3.76 (t,J=10.2 Hz, 1H), 3.51 (d, J=6.3 Hz, 2H), 3.20 (s, 3H), 3.07 (s, 3H), 2.85(dq, J=15.3, 7.1 Hz, 1H), 2.41 (dd, J=13.9, 6.3 Hz, 1H), 1.34 (d, J=16.5Hz, 15H). ¹³C NMR (75 MHz, CDCl₃) δ159.36, 158.34, 157.89, 156.59,149.39, 125.80, 105.45, 98.98, 87.28, 85.52, 84.62, 81.21, 80.16, 78.97,77.63, 63.33, 57.83, 50.97, 48.01, 47.51, 41.63, 37.96, 35.37, 31.95,30.30, 26.07, 25.99, 25.66, 23.02, 14.48.

3′-O-DTM-7-deaza-dGTP-7—SS—NH₂ (Compound G7): Compound G6 (40 mg, 53μmol), tetrabutylammonium pyrophosphate (89 mg, 160 μmol) and2-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one (22 mg, 110 μmol) weredried separately over night under high vacuum at ambient temperature.The tetrabutylammonium pyrophosphate was dissolved in dimethylformamide(DMF, 1 mL) under argon followed by addition of tributylamine (1 mL).The mixture was injected into the solution of2-chloro-4-H-1,3,2-benzodioxaphosphorin-4-one in (DMF, 2 mL) underargon. After stirring for 1 h, the reaction mixture was added to thesolution of G6 in DMF and stirred further for 1 hour at roomtemperature. Iodine solution (0.02 M iodine/pyridine/water) was theninjected into the reaction mixture until a permanent brown color wasobserved. After 10 min, water (30 mL) was added and the reaction mixturewas stirred at room temperature for an additional 2 hours. The resultingsolution was extracted with ethyl acetate. The aqueous layer wasconcentrated in vacuo to approximately 20 mL, and concentrated NH₄OH (20ml) was added and stirring continued overnight at room temperature. Theresulting mixture was concentrated under vacuum and the residue wasdiluted with 5 ml of water. The crude mixture was then purified by anionexchange chromatography on DEAE-Sephadex A-25 at 4° C. using a gradientof TEAB (pH 8.0; 0.1-1.0 M). The crude product was further purified byreverse-phase HPLC to afford G7, which was characterized by MALDI-TOFMS: calc'd for C₂₄H₄₀N₅O₁₄P₃S₄: 843.7, found: 848.0.

3′-O-DTM-7-deaza-dGTP-7—SS-Cy5 (Compound G8): To a stirred solution ofCy5—NHS ester (2 mg, 3.2 μmol) in DMF (0.2 ml),7-deaza-3′-O-DTM-dGTP-7—SS-NH₂ (compound G7, 3.0 _(t)tmol) inNaHCO₃/Na₂CO₃ buffer (pH 8.9, 0.1 M, 0.3 ml) was added. The reactionmixture was stirred at room temperature for 3 h with exclusion of light.The reaction mixture was purified by anion exchange chromatography onDEAE-Sephadex A-25 at 4° C. using a gradient of TEAB (pH 8.0; 0.1-1.0M). The crude product was further purified by reverse-phase HPLC toafford Compound G8, which was characterized by MALDI-TOF MS: calc'd forC₅₇H₇₇N₇O₂₁P₃S₆ ⁻: 1481.6, found: 1485.2.

Synthesis of 3′-O-DTM(SS)-dNTP-SS-“Anchor” Molecules. Example synthesesof 3′-O-DTM(SS)-dNTP-SS-“Anchor” are shown in FIGS. 42-45 followingprocedures similar to those for syntheses of 3′-O-DTM(SS)-dNTP-SS-Dye.Instead of using a variety of dye-NHS esters, a variety of “Anchor” NHSesters are used to couple with 3′-O-DTM(SS)-dNTP-SS-NH₂ precursorsyielding 3′-O-DTM(SS)-dNTP-SS-N₃ (FIG. 42 ), 3′-O-DTM(SS)-dNTP-SS-PBA(FIG. 43 ), 3′-O-DTM(SS)-dNTP-SS-Biotin (FIG. 44 ) and3′-O-DTM(SS)-dNTP-SS-TCO (FIG. 45 ).

Synthesis of 3′-O-DTM(SS)-dNTP-SS-“Anchor” Molecules. Example synthesesof 3′-O-DTM(SS)-dNTP-SS-“Anchor” are shown in FIGS. 42-45 followingprocedures similar to those for syntheses of 3′-O-DTM(SS)-dNTP-SS-Dye.Instead of using a variety of dye-NHS esters, a variety of “Anchor” NHSesters are used to couple with 3′-O-DTM(SS)-dNTP-SS-NH₂ precursorsyielding the3′-O-DTM(SS)-dNTP-SS-“Anchor” Molecules depicted in schemesbelow.

Synthesis of 3′-DTM-Blocked Group dNTP Analogues: General structures ofthese derivatives are shown in FIG. 36 .

1,1′-thiodialkyl(R₁—CH—R₂) is used to produce Ri,R₂ substituted3′-O-alkyl(bearing R₁R₂)thiomethyl-5′-O-(tert-butyldiphenylsilyl)nucleoside in the presence of Bz₂O₂ and 2,6-lutidine. The resultingcompounds are treated with sulfuryl chloride, potassiump-toluenethiosulfonate and corresponding thiol compounds bearing R₃, R₄and R₅ groups yielding R₁, R₂ substituted 3′-O-alkyl(bearing R₃, R₄ andR₅)dithiomethyl-5′-O-(tert-butyldiphenylsilyl) nucleoside. After removalof the 5′-O-tert-butyldiphenylsilyl protecting group with 1.0M of TBAFin THF, the resulting compound with free 5′—OH can be converted totriphosphate by using established triphosphorylation methods affordingR₁,R₂ substituted 3′-O-alkyl(with R₃, R₄ and R₅)dithiomethyl-dNTPs.

DMSO or DMSO-d6 is used to treat 5′-O-(tert-butyldiphenylsilyl)nucleoside producing3′-O-(methylthiomethyl)-5′-O-(tert-butyldiphenylsilyl) nucleoside ordeuterium substituted3′-O-methylthiomethyl-5′-O-(tert-butyldiphenylsilyl) nucleoside.Treatment of the resulting compounds with sulfuryl chloride, potassiump-tolucnethiosulfonatc, and corresponding alkylthiol (bearing R₃, R₄ andR₅ groups) will produce 3′-O-(R₃,R₄,R₅-alkyldithiomethyl)-5′-O-(tert-butyldiphenylsilyl) nucleosideor deuterium substituted 3′-O-(R₃,R₄,R₅-alkyldithiomethyl(D2)-5′-O-(tert-butyldiphenylsilyl)nucleoside. Removal of the 5′-O protecting group followed bytriphosphorylation and deprotection on the base (if applicable) willafford the final product 3′-O-(alkyl(with R₃, R₄, and R₅groups)dithiomethyl)-dNTP or deuterium substituted 3′-O-(alkyl(with R₃,R₄, and R₅ groups)thiomethyl(d2))-dNTP.

1,1′-thiodialkyl(R₁—CH—R₂) is used to produce R₁,R₂ substituted3′-O-(alkyl(bearing R₁,R₂) thiomethyl)-5′-O-(tert-butyldiphenylsilyl)nucleoside in the presence of Bz₂O₂ and 2,6-lutidine. The resultingcompounds are treated with sulfuryl chloride, potassiump-toluenethiosulfonate and corresponding thiol compounds bearing X—R₃,R₄, and Rs (X═O, S or NH) groups yielding analogs of R₁,R₂ substituted3′-O-(alkyl(with X—R₃, R₄, andR₅)dithiomethyl)-5′-O-(tert-butyldiphenylsilyl) nucleoside. Afterremoval of the 5′-O-tert-butyldiphenylsilyl protecting group with 1.0Mof TBAF in THF, the resulting compounds with free 5′-OH can be convertedto triphosphate by using established triphosphorylation methodsaffording R₁ R₂ substituted 3′-O-(alkyl(with X—R₃, R₄, andR₅)dithiomethyl)-dNTP analogs.

The resulting compounds are then treated with sulfuryl chloride,potassium p-toluenethiosulfonate and one of the corresponding thiolcompounds containing X—R₃,R₄, and R₅ groups (X═O, S, or NH) yielding3′-O-(alkyl(bearing X—R₃,R₄, and R₅groups)dithiomethyl)-5′-O-(tert-butyldiphenylsilyl) nucleoside ordeuterium substituted 3′-O-(alkyl(bearing X—R₃,R₄, and R₅groups)dithiomethyl)-5′-O-(tert-butyldiphenylsilyl) nucleoside. Afterremoval of the 5′-O protecting group with 1.0M of TBAF in THF, the 5′hydroxyl group is converted to triphosphate and the resulting compoundsare treated with ammonium hydroxide to remove the protecting group onthe base (if applicable) yielding the final products 3′-O-(alkyl(bearingX—R₃,R₄,and R₅ groups)dithiomethyl)-dNTPs or deuterium substituted3′-O-(alkyl(bearing X—R₃, R₄, and R₅ groups)dithiomethyl)-dNTPs.

The resulting coumpound is treated with sulfuryl chloride, potassiump-toluenethiosulfonate and 1,1-dimethylethylthiol producing methylsubstituted 3′-O-tert-butyldithiomethyl -5′-O-(tert-butyldiphenylsilyl)thymidine. Then the tert-butyldiphenylsilyl protecting group is removedwith 1.0M of TBAF in THF and the resulting compound is further convertedto methyl substituted 3′-O-tert-butyldithiomethyp-dTTP by usingestablished triphosphorylation methods.

The resulting coumpound is treated with sulfuryl chloride, potassiump-toluenethiosulfonate and 1,1-dimethylethylthiol producing deuteriumsubstituted3′-O-tert-butyldithiomethyl-5^(.)-O-(tert-butyldiphenylsilyl) thymidine.After removal of the tert-butyldiphenylsilyl protecting group with 1.0Mof TBAF in THF, the resulting compound is converted to deuteriumsubstituted 3-O-tert-butyldithiomethyl)-dTTP by using establishedtriphosphorylation methods.

The resulting coumpound is then treated with sulfuryl chloride,potassium p-toluenethiosulfonate and methoxymethylthiol producing3′-O-methoxymethyldithiomethyl-5′-O-(tert-butyldiphenylsilyl) thymidine.After removal of the tert-butyldiphenylsilyl protecting group with 1.0Mof TBAF in THF, the resulting compound is converted to3′-O-methoxymethyldithiomethyl-dTTP by using establishedtriphosphorylation methods.

Synthesis of Dye Labeled Binding Molecules. Synthesis of labeled bindingmolecules conjugated with fluorescent dyes is conducted by couplingcommercially available binding molecule starting materials with variousactivated dyes. Example synthesis of Rox Labeled Tetrazine, Alexa488Labeled SHA and R6G Labeled Dibenzocyclooctyne(DBCO) is shown in thebelow scheme.

Synthesis of multiple-dye conjugated binding molecules (Cy5-tetrazine asan example) is shown in the schemes below.

Synthesis of Rox-7-Cy5 labeled SHA (shown in FIG. 11 A).Cy5 labeled CPG(Glen Research) is used to start solid phase oligonucleotide synthesison a DNA synthesizer. dSpacer phosphoramidite is used as the buildingblock for seven consecutive coupling cycles, then Rox labeled dTphosphoramidite is used in the next coupling cycle. C5 amino modifierphosphoramidite is used in the last coupling cycle. After cleavage undermild conditions following the GlenResearch protocol, the amino modifiedRox-7-Cy5 product is produced and purified by HPLC. Coupling of SHA NHSester with amino modified Rox-7-Cy5 in DMSO/NaCO₃, NaHCO₃ buffer (pH8.9) will afford Rox-7-Cy5 labeled SHA.

Synthesis of Rox-3-Cy5 labeled DBCO (shown in FIG. 11 B). Cy5 labeledCPG (Glen Research) is used to start solid phase oligonucleotidesynthesis on a DNA synthesizer. dSpacer phosphoramidite is used as thebuilding block for three consecutive coupling cycles, then Rox labeleddT phosphoramidite is used in the next coupling cycle. C5 amino modifierphosphoramidite is used in the last coupling cycle. After cleavage undermild conditions following the GlenResearch protocol, the amino modifiedRox-3-Cy5 product is produced and purified by HPLC. Coupling of DBCO NHSester with amino modified Rox-3-Cy5 in DMSO/NaCO₃, NaHCO₃ buffer (pH8.9) will afford Rox-3-Cy5 labeled DBCO.

Syntheses of labeled binding molecules conjugated with fluorescent dyesvia different cleavable linkers (the structures of these molecules areshown in FIG. 15 ) are shown in FIGS. 59-65 . Synthesis of thesecompounds is achieved by coupling commercially available activated dyeswith binding molecules containing cleavable linkage moieties, which aresynthesized using commercially available materials.

The example synthesis of Streptavidin-Dimethylketal(linker)-ATTO647N isshown and the construction of the Dimethylketal linker moiety isaccomplished using a literature method.

Streptavidin is shown as the dark semi-circle (i.e.

)

Example Syntheses of 3′-O-DTM(SS)-dNTP-Linker-Dye-or-“Anchor” (FIGS. 67and 68 ) are shown in the Schemes below. Instead of using(TFA)NH-DTM(SS)-Alkyne linker, a variety of (TFA)NH-Linker-Alkyne areused to couple with 3′-O-DTM(SS)-5(7)-iodo nucleosides yielding5(7)-linker modified nucleosides, which can then be converted to5(7)-linker-NH2 modified dNTPs via triphosphorylation at the 5′—OH.Lastly, either dye NHS esters or “anchor”—NHS esters can be used tocouple with 3′-O-DTM(SS)-5(7)-linker-NH₂ modified dNTPs affording avariety of 3′-O-DTM(SS)-dNTP-Linker-Dye or—“Anchor”.

Scheme 34. Synthesis of 3′-O-DTM-dCTP-5-Nitrobenzyl-Rox is shown in FIG.37 .

Scheme 35. Synthesis of 3′-O-DTM-dUTP-5-Allyl-Rox is shown in FIG. 38 .

Scheme 36. More generally, synthesis of 3′-O-DTM-dNTP-Nitrobenzyl-R, inwhich R is either a dye or an anchor, is shown in FIG. 39 .

Scheme 37. Synthesis of 3′-O-DTM-dNTP-Allyl-R is shown in FIG. 40 .

Schemes 38 and 39. The synthesis of either dye or anchor labeled3′-O-DTM(SS)-dNTPs via Azo and Dde linkers respectively in FIGS. 41 and42 .

Example 4 Long DNA synthesis Using 3′-O-alkyl-dithiomethyl-dNTPs

DNA synthesis using nucleoside phosphoramidite chemistry is a standardprocess and used for most of the custom DNA synthesis needs. However,the synthesis is limited to short length (<120 nucleotides) and it isimpractical to synthesize DNA of >200 nucleotides in length. Thesynthesis also involves the use and generation of toxic by-products andthe disposal of such toxic waste increase the cost of DNA synthesis(LeProust et al., Nucleic Acids Res. (2010) 38(8), 2522-2540).

The invention provides improved methods for DNA synthesis using3′-O-DTM-dNTPs nucleotides of the present invention as described belowand systematically in the above scheme. These nucleotides can be used inboth template-dependent (using DNA polymerase) and template-independentsynthesis of polynucleotides (DNA) by using a terminal deoxynucleotidyltransferase enzyme. These nucleotides carry a chemically cleavabledisulfide linkage at the 3′-O-position which pause the synthesis aftersingle nucleotide addition. After mild treatment with THP or TCEP, the3′-blocking group is cleaved and the generation of free 3′—OH groupresults in the addition of next nucleotide.

Terminal deoxynucleotidyl transferase enzyme is known to incorporatenatural and modified nucleotides at the 3′-end of the polynucleotides intemplate independent manner. Various attempts have been made to use TdTfor controlled de novo single-stranded DNA synthesis (Ud-Dean S.M.M.Syst. Synth Biol. (2009) 2, 67-73, U.S. Pat. Nos. 5,763,594 and8,808,989). A reversible nucleoside triphosphate is necessary to preventuncontrolled addition of dNTPs to the 3′-end of a growing DNA strand.However the efficiency of TdT to incorporate 3′-O-modified nucleotidesis very limited (WO 2016/128731 A1). A mutant TdT enzyme may also beused to incorporate 3′-O-reversibly terminated nucleotides of thisinvention.

Thus, present invention provides the method of making long DNA comprisesthe steps of:

a) an initiator sequence bound to solid support;

b) adding a 3′-O-alkyl-dithiomethyl dNTP (3′-O-DTM-dNTP) to saidinitiator sequence in the presence of terminal deoxynucleotidyltransferase (TdT);

c) removal of reagents from the initiator sequence;

d) cleavage of the 3′-O-blocking group from the extended sequence in thepresence of cleaving agent such as THP or TCEP;

e) removal of cleaving reagents;

f) repeat the sequence b-e.

The present method also provides the kit for nucleic acid synthesiscomprises a mutant TdT, an initiator sequence and a set of four or more3′-O-reversibly blocked dNTPs, buffers, cleaving agent, and instructionsfor the use of the kit for DNA synthesis.

The DNA synthesis can also be carried out using a template/primer, DNApolymerase and four 3′-O— reversibly blocked dNTPs of this invention.This will result in the formation of double stranded DNA of definedlength which can be denatured and separated as single stranded DNA.

Example 5 Synthetic Methods

General synthetic methods for analogues of 3′-O-Cleavabl eG roup-dNTP-SS-Label is shown. Starting from 5(7) iodide substitutednucleosides, the 5′-OH and amino groups on the base are protected. Thenthe 3′-OH is converted to 3′-O—R⁶ using various established syntheticmethods. The resulting compounds are coupled with a(TFA)NH-DTM(SS)-alkyne building block via Sonogashira coupling yielding5(7)-(TFA)NH-SS-nucleosides. After removal of the 5¹—Oprotecting group,the 3′-O—R⁶-5(7)-SS-nucleosides are converted to 3′-O-R⁶-5(7)-SS-dNTPsusing the established triphosphorylation method. Further deprotection ofthe amino group affords 3′-O—R⁶-5(7)-NH₂—SS-dNTPs. The resulting3′-O—R⁶-5(7)-NH₂—SS-dNTP precursors are then reacted with Label (Anchoror Dye)-NHS esters to produce 3′-O-CleavableGroup(R⁶)-dNTP-SS-Labc1(R),where R refers to Dye or Anchor molecules.

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1. A compound of the formula: Wherein

B is a base; L¹ is covalent linker; L² is covalent linker; R³ is —OH,monophosphate, polyphosphate or a nucleic acid; R^(4A) is hydrogen,—CH₃, —CX¹ ₃, —CHX¹ ₂, —CH₂X¹, —CN, -Ph, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl,substituted or unsubstituted aryl, or substituted or unsubstitutedheteroaryl; R^(4B) is hydrogen. —CH₃, —CX² ₃, —CHX² ₂, —CH₂X², —CN, -Ph,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted heterocycloalkyl, substituted or unsubstituted aryl, orsubstituted or unsubstituted heteroaryl; R⁵ is a detectable label oranchor moiety; R⁶ is hydrogen or a polymerase-compatible cleavablemoiety and the polymerase-compatible cleavable moiety is —CH₂—CH═CH₂,—CH₂N₃ or

R^(8A) is hydrogen: R^(8B) is hydrogen; R⁹, R¹⁰, and R¹¹ are eachindependently hydrogen or unsubstituted alkyl; R⁷ is hydrogen or—OR^(7A), wherein R^(7A) is hydrogen; and X¹, X², X³, and X⁴ areindependently halogen.
 2. The compound of claim 1, wherein B is adivalent cytosine or a derivative thereof, a divalent guanine or aderivative thereof, a divalent adenine or a derivative thereof, divalentthymine or a derivative thereof, a divalent uracil or a derivativethereof, a divalent hypoxanthine or a derivative thereof, a divalentxanthine or a derivative thereof, a deaza-adenine or a derivativethereof, a deaza-guanine or a derivative thereof, a deaza-hypoxanthineor a derivative thereof, a divalent 7-methylguanine or a derivativethereof, a divalent 5,6-dihydrouracil or a derivative thereof, adivalent 5-methylcytosine or a derivative thereof, or a divalent5-hydroxymethylcytosine or a derivative thereof.
 3. The compound ofclaim 1, wherein B is


4. The compound of claim 1, wherein L¹ isL^(1A)-L^(1B)-L^(1C)-L^(1D)-L^(1E); and L^(1A), L^(1B), L^(1C), L^(1D)and L^(1E) are independently a bond, substituted or unsubstitutedalkylene, substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene; wherein at least one ofL^(1A), L^(1B), L^(1C), L^(1D) and L^(1E) is not a bond.
 5. The compoundof claim 1, wherein L¹ is L^(1A)-L^(1B)-L^(1C)-L^(1D)-L^(1E); andL^(1A), L^(1B), L^(1C), L^(1D) and L^(1E) are independently a bond,substituted or unsubstituted C₁-C₈ alkylene, substituted orunsubstituted 2 to 8 membered heteroalkylene, substituted orunsubstituted C₃-C₈ cycloalkylene, substituted or unsubstituted 3 to 8membered heterocycloalkylene, substituted or unsubstituted C₆-C₁₀arylene, or substituted or unsubstituted 5 to 10 membered heteroarylene;wherein at least one of L^(1A), L^(1B), L^(1C), L^(1D) and L^(1E) is nota bond.
 6. (canceled)
 7. A method of incorporating a nucleotide analogueinto a primer, the method comprising combining a polymerase, a primerhybridized to nucleic acid template and a nucleotide analogue within areaction vessel and allowing said polymerase to incorporate saidnucleotide analogue into said primer thereby forming an extended primer,wherein said nucleotide analogue is of the structure of claim
 1. 8.(canceled)
 9. The compound of claim 1, wherein L² isL^(2A)-L^(2B)-L^(2C)-L^(2D)-L^(2E); and L^(2A), L^(2B), L^(2C), L^(2D)and L^(2E) are independently a bond, substituted or unsubstitutedalkylene, substituted or unsubstituted heteroalkylene, substituted orunsubstituted cycloalkylene, substituted or unsubstitutedheterocycloalkylene, substituted or unsubstituted arylene, orsubstituted or unsubstituted heteroarylene; wherein at least one ofL^(2A), L^(2B), L^(2C), L^(2D) and L^(2E) is not a bond.
 10. Thecompound of claim 1, wherein L² is L^(2A)-L^(2B)-L^(2C)-L^(2D)-L^(2E);and L^(2A), L^(2B), L^(2C), L^(2D) and L^(2E) are independently a bond,—NN—, —NHC(O)—, —C(O)NH—, substituted or unsubstituted C₁-C₂₀ alkylene,substituted or unsubstituted 2 to 20 membered heteroalkylene,substituted or unsubstituted C₃-C₂₀ cycloalkylene, substituted orunsubstituted 3 to 20 membered heterocycloalkylene, substituted orunsubstituted C₆-C₂₀ arylene, or substituted or unsubstituted 5 to 20membered heteroarylene wherein at least one of L^(2A), L^(2B), L^(2C),L^(2D), and L^(2E) is not a bond.
 11. The compound of claim 1, whereinR³ is triphosphate.
 12. The compound of claim 1, wherein R^(4A) andR^(4B) are each independently hydrogen or —CH₃.
 13. The compound ofclaim 1, wherein R⁵ is a fluorescent dye.
 14. The compound of claim 1,wherein R⁷ is hydrogen.
 15. The compound of claim 1, wherein R⁶ is—CH₂—CH═CH₂ or —CH₂N₃.
 16. The compound of claim 1, wherein the compoundhas the formula:

wherein m is an integer from 1 to
 4. 17. The compound of claim 16,wherein in is 1 or
 4. 18. The compound of claim 1, wherein the compoundhas the formula:


19. The compound of claim 1, wherein the compound has the formula:


20. A method for sequencing a nucleic acid, comprising: (i)incorporating in series with a nucleic acid polymerase, within areaction vessel, one of four different labeled nucleotide analogues intoa primer to create an extension strand, wherein said primer ishybridized to said nucleic acid and wherein each of the four differentlabeled nucleotide analogues comprise a unique detectable label; (ii)detecting said unique detectable label of each incorporated nucleotideanalogue, so as to thereby identify each incorporated nucleotideanalogue in said extension strand, thereby sequencing the nucleic acid;and wherein each of said four different labeled nucleotide analogues areof the structure of claim 1, wherein in the first of said four differentlabeled nucleotide analogues, B is a thymidine or uridine hybridizingbase; in the second of said four different labeled nucleotide analogues,B is an adenosine hybridizing base; in the third of said four differentlabeled nucleotide analogues, B is a guanosine hybridizing base; and inthe fourth of said four different labeled nucleotide analogues, B is acytosine hybridizing base.
 21. A method for sequencing a nucleic acid,comprising: (i) incorporating in series with a nucleic acid polymerase,within a reaction vessel, one of four different labeled nucleotideanalogues into a primer to create an extension strand, wherein saidprimer is hybridized to said nucleic acid and wherein each of the fourdifferent labeled nucleotide analogues comprise a unique detectablelabel; (ii) detecting said unique detectable label of each incorporatednucleotide analogue, so as to thereby identify each incorporatednucleotide analogue in said extension strand, thereby sequencing thenucleic acid; and wherein each of said four different labeled nucleotideanalogues a of the structure of claim 16, wherein in the first of saidfour different labeled nucleotide analogues, B is a thymidine or uridinehybridizing base; in the second of said four different labelednucleotide analogues, B is an adenosine hybridizing base; in the thirdof said four different labeled nucleotide analogues, B is a guanosinehybridizing base; and in the fourth of said four different labelednucleotide analogues, B is a cytosine hybridizing base.
 22. A method forsequencing a nucleic acid, comprising: (i) incorporating in series witha nucleic acid polymerase, within a reaction vessel, one of fourdifferent labeled nucleotide analogues into a primer to create anextension strand, wherein said primer is hybridized to said nucleic acidand wherein each of the four different labeled nucleotide analoguescomprise a unique detectable label; (ii) detecting said uniquedetectable label of each incorporated nucleotide analogue, so as tothereby identify each incorporated nucleotide analogue in said extensionstrand, thereby sequencing the nucleic acid; and wherein each of saidfour different labeled nucleotide analogues are of the structure ofclaim 19, wherein in the first of said four different labeled nucleotideanalogues, B is a thymidine or uridine hybridizing base; in the secondof said four different labeled nucleotide analogues, B is an adenosinehybridizing base; in the third of said four different labeled nucleotideanalogues, B is a guanosine hybridizing base; and in the fourth of saidfour different labeled nucleotide analogues, B is a cytosine hybridizingbase.